JP7348980B2 - Soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptides and uses thereof - Google Patents

Description

The invention features soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptides and compositions thereof. The invention also features methods of treating skeletal growth retardation disorders, such as achondroplasia.

Fibroblast growth factor receptor 3 (FGFR3) is a member of the fibroblast growth factor (FGFR) family in which there is high amino acid sequence conservation among family members. Members of the FGFR family are differentiated by both ligand binding affinity and tissue distribution. A full-length FGFR polypeptide contains an extracellular domain (ECD), a hydrophobic transmembrane domain, and a cytoplasmic tyrosine kinase domain. The ECD of the FGFR polypeptide interacts with fibroblast growth factors (FGFs) to mediate downstream signaling, which ultimately influences cell differentiation. In particular, activation of FGFR3 protein plays a role in bone development by inhibiting cartilage proliferation in the growth plate and limiting bone elongation.

Gain-of-function mutations in FGFR3 have been linked to several types of human skeletal growth retardation disorders, such as achondroplasia, thanatophoric osteodysplasia type I (TDI), and thanatophoric osteodysplasia type II ( TDII), severe achondroplasia with growth retardation and acanthosis nigricans (SADDAN), achondroplasia, and craniosynostosis syndromes (e.g., Muenke syndrome, Crouzon syndrome, and Crouzonodermaskeletal syndrome) ) is known to cause Loss-of-function point mutations in FGFR3 are also known to cause skeletal growth retardation disorders, such as Arthrodactyly, Tallness, and Hearing Loss Syndrome (CATSHL). Achondroplasia is the most common form of shortened limb dwarfism and is characterized by disproportionate limb shortness and relative macrocephaly. Approximately 97% of achondroplasias are caused by a single point mutation in the gene encoding FGFR3, in which a glycine residue is replaced by an arginine residue at position 380 of the FGFR3 amino acid sequence. Upon ligand binding, the mutation reduces clearance of the receptor/ligand complex, resulting in long-term intracellular signaling. This long-term FGFR3 signaling inhibits cartilage growth plate proliferation and differentiation, resulting in impaired endochondral bone growth.

There is a need for improved therapeutics that target dysfunctional FGFR3 to treat skeletal growth retardation disorders, such as achondroplasia.

The present invention relates to soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptides and their uses, such as skeletal growth retardation disorders (e.g., cartilage) in patients (e.g., humans, particularly infants, children, or young adults). Aplasia). In particular, the sFGFR3 polypeptides of the invention are characterized, for example, by a deletion of amino acids 289-400 of the amino acid sequence of the wild-type FGFR3 polypeptide (e.g., SEQ ID NO: 5 or 32 amino acid sequences): sFGFR3_Del4 (sFGFR3_Del4-C253S; SEQ ID NO: 2) containing an amino acid substitution of a cysteine residue by a serine residue at position 253 and sFGFR3_Del4 (sFGFR3_Del4-D3) containing an extended Ig-like C2 type domain 3. ; SEQ ID NO: 33) and variants thereof, such as the sFGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 4. Additionally, the sFGFR3 polypeptide of the invention may include a signal peptide, such as an sFGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34.

A first aspect of the invention provides at least 90% amino acid sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (eg, 100%) sequence identity). In particular, the polypeptide lacks the signal peptide (e.g., amino acids 1-22 of SEQ ID NO: 32) and transmembrane domain (e.g., amino acids 367-399 of SEQ ID NO: 32) of FGFR3, and (i) is less than 500 amino acids in length ( (ii) less than 200 contiguous amino acids (e.g., 175, 150, 125, 100, 75, 50, 40, 30, 20 , 15 or fewer contiguous amino acids); and/or (iii) lacks the tyrosine kinase domain of FGFR3. The sFGFR3 polypeptide has at least 90%, 92%, 95%, 97% or 99% of the intracellular domain of FGFR3, e.g., amino acid residues 423-435 of SEQ ID NO: 32 or amino acid residues 423-435 of SEQ ID NO: 32. may also include amino acid sequences having sequence identity of . In particular, the polypeptide comprises an amino acid sequence that has at least 92%, 95%, 97%, 99% or 100% sequence identity with SEQ ID NO: 33 (e.g., the polypeptide comprises or is derived from SEQ ID NO: 33). Become). The sFGFR3 polypeptide may also include a signal peptide (e.g., the signal peptide has the amino acid sequence of SEQ ID NO: 6 or 35 or at least 92%, 95%, 97% or 99% sequence identity with SEQ ID NO: 6 or 35). amino acid sequence). For example, an sFGFR3 polypeptide can have an amino acid sequence that has at least 92%, 95%, 97%, 99% or 100% sequence identity with SEQ ID NO: 34 (e.g. (including or consisting of). The sFGFR3 polypeptide may also have a heterologous signal peptide (eg, the polypeptide includes a heterologous signal peptide having the amino acid sequence of SEQ ID NO: 35).

A second aspect of the invention provides at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) to the amino acid sequence of SEQ ID NO: 1. , 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), wherein the cysteine residue at position 253 of SEQ ID NO: 1 is removed. Features sFGFR3 polypeptides that further include amino acid substitutions. For example, the cysteine residue at position 253 is replaced by a serine residue or another conservative amino acid substitution, such as alanine, glycine, proline, or threonine. In particular, the sFGFR3 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:2. For example, the sFGFR3 polypeptide can be an isolated sFGFR3 polypeptide. The sFGFR3 polypeptide may also include a signal peptide (e.g., the signal peptide has the amino acid sequence of SEQ ID NO: 6 or 35 or at least 92%, 95%, 97% or 99% sequence identity with SEQ ID NO: 6 or 35). amino acid sequence). For example, sFGFR3 can have an amino acid sequence that has at least 92%, 95%, 97%, 99% or 100% sequence identity with SEQ ID NO: 18 (e.g., the sFGFR3 polypeptide comprises SEQ ID NO: 18. or consisting of). The sFGFR3 polypeptide may also have a heterologous signal peptide (eg, the polypeptide includes a heterologous signal peptide having the amino acid sequence of SEQ ID NO: 35).

A third aspect of the invention provides at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%) to the amino acid sequence of SEQ ID NO: 1. , 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), wherein all or a fragment of the amino acid sequence of SEQ ID NO: 3 (e.g., at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity), the domain having an amino acid sequence of SEQ ID NO. The sFGFR3 polypeptide is characterized by an insertion between amino acid residues 288 and 289. For example, a domain can include an amino acid sequence that has at least 85%, 90%, 92%, 95%, 97% or 99% sequence identity with the amino acid sequence of SEQ ID NO: or consisting of the amino acid sequence). Optionally, the sFGFR3 polypeptide contains a serine residue or another conservative amino acid substitution, e.g., cysteine at position 253 of SEQ ID NO: 1 and/or position 28 of SEQ ID NO: 3, such as alanine, glycine, proline, or threonine. Contains amino acid substitutions of residues. In particular, the sFGFR3 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:4. For example, the sFGFR3 polypeptide can be an isolated sFGFR3 polypeptide.

The first or second of the present invention comprises a nucleic acid sequence having at least 85%, 90%, 92%, 95%, 97% or 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 20, 21, 36 or 37 , or a polynucleotide (e.g., an isolated polynucleotide) encoding the sFGFR3 polypeptide of the third aspect (e.g., the polynucleotide comprises a nucleic acid of SEQ ID NO: 20, 21, 36, or 37, or consisting of). The invention also features vectors (eg, isolated vectors) that include polynucleotides, such as plasmids, artificial chromosomes, viral vectors, or phage vectors. Additionally, the invention features host cells (eg, isolated host cells) that include the polynucleotide, such as HEK293 cells or CHO cells.

The present invention provides a composition comprising the sFGFR3 polypeptide of the first, second or third aspect of the invention or a polynucleotide encoding the sFGFR3 polypeptide of the first, second or third aspect of the invention. It is characterized by Additionally, vectors or host cells containing polynucleotides encoding sFGFR3 polypeptides can be formulated in compositions. The composition may further include a pharmaceutically acceptable excipient, carrier, or diluent. Compositions comprising sFGFR3 polypeptides, polynucleotides, or vectors may be formulated for administration at a dose of about 0.002 mg/kg to about 30 mg/kg, such as about 0.001 mg/kg to about 10 mg/kg. Can be done. Compositions containing host cells can be formulated for administration at doses of about 1×10 ³ cells/mL to about 1×10 ¹² cells/mL. The compositions can be administered once a day, once a week, or once a month, such as 7 times a week, 6 times a week, 5 times a week, 4 times a week, 3 times a week, 2 times a week, once a week, It can be formulated for biweekly or monthly administration. For example, compositions containing sFGFR3 polypeptides, polynucleotides, or vectors are formulated for once or twice weekly administration at doses of about 0.25 mg/kg to about 10 mg/kg. The compositions may be formulated for parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or intraperitoneal), enteral, or topical administration. can. Preferably, the composition is formulated for subcutaneous administration. The invention also features a pharmaceutical product that includes one or more of the above compositions.

The present invention also provides for administering to a patient an effective amount of the sFGFR3 polypeptide of the first, second or third aspect of the invention, a polynucleotide encoding the sFGFR3 polypeptide, a vector containing the polynucleotide, a polynucleotide or a vector. in a patient (e.g., human) having a skeletal growth retardation disorder (e.g., achondroplasia), comprising administering a host cell containing the polypeptide, polynucleotide, vector, or composition containing the host cell. Features a method of delivering an sFGFR3 polypeptide to a tissue (eg, skeletal tissue).

A fourth aspect of the present invention provides a polynucleotide encoding the polypeptide or polypeptide of the first, second, or third aspect of the present invention, a vector containing the polynucleotide, or a host containing the polynucleotide or vector. A method of treating a skeletal growth retardation disorder (e.g., FGFR3-associated skeletal disease) in a patient (e.g., human) comprising administering a cell, or a composition comprising a polypeptide, polynucleotide, vector, or host cell. Features. FGFR3-related skeletal diseases include achondroplasia, thanatophoric osseous dysplasia type I (TDI), thanatophoric osseous dysplasia type II (TDII), severe achondroplasia with growth retardation and acanthosis nigricans. syndrome (SADDAN), achondrohypoplasia, craniosynostosis syndrome (eg, Muenke syndrome, Crouzon syndrome, and Crouzon dermatoskeletal syndrome), and syndrome of tall stature and hearing loss (CATSHL). In particular, a skeletal growth retardation disorder is achondroplasia.

FGFR3-related skeletal diseases can be caused by expression in a patient of constitutively active FGFR3, eg, amino acid substitution of a glycine residue by an arginine residue at position 380 of SEQ ID NO: 5 or 32. In particular, the patient may have been diagnosed (eg, prior to treatment) with a skeletal growth retardation disorder. For example, patients may suffer from shortened limbs, short torso, bowlegs, agitated gait, cranial dysmorphia, cloverleaf cranial craniosynostosis, craniosynostosis, worm bones, hand malformations, foot deformities, hitchhiker's thumb, and thoracic deformities. The patient exhibits one or more symptoms of a skeletal growth retardation disorder selected from the group consisting of: or a composition containing a polypeptide, polynucleotide, vector, or host cell) that exhibits an amelioration of one or more symptoms of a skeletal growth retardation disorder. Additionally, the patient may not have previously been administered an sFGFR3 polypeptide. For example, a patient can be an infant, child, young adult, or adult.

For example, the polypeptide is administered to a patient at a dose of about 0.002 mg/kg to about 30 mg/kg (eg, a dose of about 0.001 mg/kg to about 10 mg/kg). The polypeptide may be administered to the patient at least once a day, at least once a week (e.g., twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week), once every two weeks. As above, it can be administered once a month or more, or once a year or more. For example, the polypeptide is administered to the patient at a dose of about 0.25 mg/kg to about 30 mg/kg at least about once or twice a week (eg, the polypeptide is administered to the patient at a dose of about 2.5 mg/kg or (administered once or twice a week at a dose of approximately 10 mg/kg). A polypeptide can be administered to a patient in a composition that includes a pharmaceutically acceptable excipient, carrier, or diluent. Polypeptides can be administered to a patient parenterally (eg, subcutaneously, intravenously, intramuscularly, intraarterially, intrathecally, or intraperitoneally), enterally, or locally. Preferably, the composition is administered to the patient by subcutaneous injection. Additionally, the polypeptides include fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 18 (FGF18), fibroblast growth It may bind factor 19 (FGF19), or fibroblast growth factor 21 (FGF21). In particular, binding can be characterized by an equilibrium dissociation constant (K _d ) of about 0.2 nM to about 20 nM; , about 5 nm, about 6 nm, _about 7 nm, about 8 nm, about 9 nm, or about 10 nm). The polypeptide may exhibit greater binding affinity for FGF1, FGF2, FGF9, and FGF18 relative to the polypeptide's binding affinity for FGF19 and FGF21.

The polypeptide is administered for about 2 hours to about 25 hours (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, It may have an in vivo half-life of 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 25 hours). Preferably, administration of the polypeptide results in one or more or all of the following: increasing survival in the patient, improving locomotion in the patient, improving abdominal breathing in the patient, increasing body length and/or bone length in the patient, Improving the cranial ratio of the patient and/or restoring the shape of the foramen magnum in the patient, for example, relative to untreated patients (eg, untreated achondroplasia patients).

The invention also provides the steps of culturing the host cells (e.g., CHO cells or HEK293 cells) in a culture medium under conditions suitable to cause expression of the sFGFR3 polypeptide, and recovering the sFGFR3 polypeptide from the culture medium. A method of producing the sFGFR3 polypeptide of the first, second, or third aspect of the invention, comprising: In particular, recovery involves chromatography, such as affinity chromatography (eg ion exchange chromatography or anti-FLAG chromatography, eg immunoprecipitation) or size exclusion chromatography.

A fifth aspect of the invention provides that the polypeptide (or polynucleotide encoding the polypeptide, polynucleotide) of the first, second or third aspect of the invention is used to treat a skeletal growth retardation disorder in a patient. a vector containing a polypeptide, a polynucleotide or a host cell containing a vector, or a composition containing a polypeptide, polynucleotide, vector, or host cell). In particular, sFGFR3 polypeptides include fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 18 (FGF18), It may bind to growth factor 19 (FGF19) or fibroblast growth factor 21 (FGF21).

A sixth aspect of the invention provides at least 85% sequence identity (e.g., at least 86%, 87%, sFGFR3 polypeptide comprising an amino acid sequence having a sequence identity of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (or a polynucleotide encoding a polypeptide, a vector containing the polynucleotide, a host cell containing the polynucleotide or vector, or a composition containing the polypeptide, polynucleotide, vector, or host cell) that The sFGFR3 polypeptide further comprises an amino acid substitution that removes the cysteine residue at position 253 of number 1. For example, the cysteine residue at position 253 is replaced with a serine residue, or another conservative amino acid substitution, such as alanine, glycine, proline, or threonine. In particular, the sFGFR3 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:2. For example, the sFGFR3 polypeptide can be an isolated sFGFR3 polypeptide. Furthermore, sFGFR3 polypeptides are associated with fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 18 (FGF18), It may bind to growth factor 19 (FGF19) or fibroblast growth factor 21 (FGF21).

A seventh aspect of the invention provides at least 85% sequence identity (e.g., at least 86%, 87%, sFGFR3 polypeptide comprising an amino acid sequence having a sequence identity of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (or a polynucleotide encoding a polypeptide, a vector containing the polynucleotide, a host cell containing the polynucleotide or vector, or a composition containing the polypeptide, polynucleotide, vector, or host cell) that at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) further comprising a domain characterized by the sFGFR3 polypeptide inserted between amino acid residues 288 and 289 of SEQ ID NO:1. For example, a domain can include an amino acid sequence that has at least 85%, 90%, 92%, 95%, 97% or 99% sequence identity with the amino acid sequence of SEQ ID NO: or consisting of the amino acid sequence). Optionally, the sFGFR3 polypeptide contains a serine residue at position 253 of SEQ ID NO: 1 and/or a cysteine residue at position 28 of SEQ ID NO: 3 or another conservative amino acid substitution, e.g., a cysteine residue with alanine, glycine, proline, or threonine. Includes amino acid substitutions of groups. In particular, the sFGFR3 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:4. For example, the sFGFR3 polypeptide can be an isolated sFGFR3 polypeptide. Additionally, sFGFR3 polypeptides are associated with fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 18 (FGF18), It may bind to growth factor 19 (FGF19) or fibroblast growth factor 21 (FGF21).

The use of the fifth, sixth or seventh aspect is also characterized by the administration of the polynucleotide, vector, host cell or composition of the first, second or third aspect of the invention. The sFGFR3 polypeptide of the sixth aspect of the invention comprises a nucleic acid sequence having at least 85%, 90%, 92%, 95%, 97% or 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 20 or 36. may be encoded by a polynucleotide (eg, the polynucleotide comprises or consists of the nucleic acid of SEQ ID NO: 20 or 36). The sFGFR3 polypeptide of the fifth or seventh aspect of the invention is a nucleic acid having at least 85%, 90%, 92%, 95%, 97% or 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 21 or 37. (eg, the polynucleotide comprises or consists of the nucleic acid of SEQ ID NO: 21 or 37).

The skeletal growth retardation disorder of the fifth, sixth or seventh aspect of the invention is any FGFR3-related skeletal disease, such as achondroplasia, TDI, TDII, severe cartilage with growth retardation and acanthosis nigricans. It can be SADDAN, achondroplasia, craniosynostosis syndrome (eg, Muenke syndrome, Crouzon syndrome, and Crouzon dermatoskeletal syndrome), or CATSHL. In particular, a skeletal growth retardation disorder is achondroplasia. FGFR3-associated skeletal diseases can be caused by expression in a patient of constitutively active FGFR3, e.g., constitutively active FGFR3 comprises an amino acid substitution of a glycine residue by an arginine residue at position 380 of SEQ ID NO: 5. .

The patient (eg, human) of the fifth, sixth, or seventh aspect of the invention may be one who has been diagnosed (eg, prior to treatment) with a skeletal growth retardation disorder. Patients presented with a group consisting of shortened limbs, short torso, bowlegs, agitated gait, cranial dysmorphia, cloverleaf skull, craniosynostosis, worm bones, hand anomalies, foot anomalies, hitchhiker's thumb, and thoracic anomalies. may exhibit one or more symptoms of selected skeletal growth retardation disorders (eg, achondroplasia). As a result of the method, a patient may exhibit improvement in one or more symptoms of a skeletal growth retardation disorder after administration of an sFGFR3 polypeptide. Additionally, administration of the sFGFR3 polypeptide may increase the patient's survival rate and/or restore the shape of the patient's foramen magnum. Patients can be infants, children, young adults, or adults. Additionally, the patient contains a sFGFR3 polypeptide (or a polynucleotide encoding the polypeptide, a vector containing the polynucleotide, a host cell containing the polynucleotide or vector, or a polypeptide, polynucleotide, vector, or host cell containing the polynucleotide). composition) may not have been previously administered.

The sFGFR3 polypeptide, polynucleotide, or vector of the fifth, sixth, or seventh aspect of the invention may be administered to a patient (eg, a human) at a dose of about 0.002 mg/kg to about 30 mg/kg, such as about 0. 001 mg/kg to about 10 mg/kg. A composition comprising a host cell of the fourth or fifth aspect of the invention is administered to a patient (e.g. a human) at a dose of about 1 x 10 ³ cells/mL to about 1 x 10 ¹² cells/mL. can be administered. For example, the sFGFR3 polypeptide, polynucleotide, vector, or host cell is administered to a patient at least once a day, at least once a week, at least once a month, or at least once a year (e.g., the sFGFR3 polypeptide is administered to a patient at least once a day, at least once a week, at least once a month, or at least once a year) (administered to patients 7 times a week, 6 times a week, 5 times a week, 4 times a week, 3 times a week, 2 times a week, once every two weeks, or once a month). In particular, the sFGFR3 polypeptide is administered to the patient at a dose of about 0.25 mg/kg to about 10 mg/kg once or twice a week. The sFGFR3 polypeptide can be administered to a patient in a composition that includes a pharmaceutically acceptable excipient, carrier, or diluent. For example, the composition is administered parenterally (eg, subcutaneously, intravenously, intramuscularly, intraarterially, intrathecally, or intraperitoneally), enterally, or locally to a patient. In particular, the composition is administered to the patient by subcutaneous injection.

The present invention provides that by deleting a portion of the signal peptide, transmembrane domain, and intracellular domain from the FGFR3 polypeptide (e.g., to produce a polypeptide having the amino acid sequence of SEQ ID NO: 33), The first aspect features a method of producing the sFGFR3 polypeptide. In particular, the intracellular domain consists of amino acid residues 436-806 of SEQ ID NO:32. The invention also provides a second method of the invention by introducing an amino acid substitution that removes the cysteine residue at position 253 of SEQ ID NO: 1 (e.g., to produce a polypeptide having the amino acid sequence of SEQ ID NO: 2). Embodiments feature methods of producing sFGFR3 polypeptides. For example, the cysteine residue at position 253 is replaced by a serine residue or another conservative amino acid substitution, such as alanine, glycine, proline, or threonine.

The invention also relates to the sFGFR3 polypeptide of the first, second or third aspect of the invention (e.g. a polypeptide having an amino acid sequence of SEQ ID NO: 2, 4 or 33), the first, second or third aspect of the invention. 2 or the third aspect (e.g. a polynucleotide having the nucleic acid sequence of SEQ ID NO: 20, 21, 36 or 37), the vector of the first, second or third aspect of the invention (e.g. , a plasmid, an artificial chromosome, a viral vector or a phage vector) or a host cell (e.g. a HEK293 cell or a CHO cell) of the first, second or third aspect of the invention, optionally comprising: , featuring a kit that includes instructions for using the kit.

DEFINITIONS As used herein, "a" and "an" mean "at least one" or "one or more" unless otherwise indicated. Additionally, the singular forms "a,""an," and "the" include plural referents unless the context clearly dictates otherwise.

"About" as used herein refers to an amount that is ±10% of the quoted value, preferably ±5% of the quoted value, more preferably ±2% of the quoted value. For example, the term "about" can be used to modify any dosage or range recited herein by ±10% of the recited value or range endpoint.

The term "domain" refers to a conserved region of amino acid sequence of a polypeptide (eg, a FGFR3 polypeptide) that has identifiable structure and/or function within the polypeptide. Domains can vary in length, for example, from about 20 amino acids to about 600 amino acids. Exemplary domains include the immunoglobulin domains of FGFR3 (eg, Ig-like C2 type domain 1, Ig-like C2 type domain 2, and Ig-like C2 type domain 3).

The term "dose" refers to the desired therapeutic effect (e.g., a skeletal growth retardation disorder, e.g., achondroplasia) when the active agent is administered to a patient (e.g., a patient with a skeletal growth retardation disorder, e.g., achondroplasia). sFGFR3 polypeptide or a variant thereof, eg, a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33). A dosage can be defined in terms of a defined amount of active agent or a defined amount that is tied to a particular frequency of administration. Dosage forms include the sFGFR3 polypeptide or fragment thereof in association with any suitable pharmaceutical excipient, carrier, or diluent.

The terms "effective amount," "amount effective for," and "therapeutically effective amount" refer to sufficient sFGFR3 polypeptide to produce a desired result, e.g., treatment of a skeletal growth retardation disorder (e.g., achondroplasia). Refers to the amount of vector encoding sFGR3 and/or sFGFR3 composition.

The terms "extracellular domain" and "ECD" refer to the portion of the FGFR3 polypeptide that extends beyond the transmembrane domain into the extracellular space. ECD mediates the binding of FGFR3 to one or more fibroblast growth factors (FGFs). For example, the ECD includes Ig-like C2 type domains 1-3 of the FGFR3 polypeptide. In particular, the ECD comprises the Ig-like C2 type domain 1 of a wild-type (wt) FGFR3 polypeptide (e.g., amino acids 36-88 of a wt FGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 5 (mature FGFR3 protein without a signal sequence)). or amino acids 57-110 of a wtFGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 32 (precursor FGFR3 protein with a signal sequence), Ig-like C2-type domain 2 of a wild-type (wt) FGFR3 polypeptide (e.g., SEQ ID NO: 5 or amino acids 161-245 of the wtFGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 32), and Ig-like C2 type domain 3 of the wtFGFR3 polypeptide (e.g., or amino acids 268-310 of a wtFGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 32). The ECD of FGFR3 may also include a fragment of wild-type FGFR3Ig-like C2-type domain 3, such as aa 247-288 of SEQ ID NO: 1, which may include, for example, a serine residue at position 253 of SEQ ID NO: 253 or another conservative amino acid substitution, such as , alanine, glycine, proline, or threonine) for cysteine residues (eg, aa 247-288 of SEQ ID NO: 2). Additionally, the ECD can include, for example, the Ig-like C2 type domain 3 of aa 247-335 of SEQ ID NO: 4. Thus, exemplary ECDs of FGFR3 polypeptides include, for example, polypeptides having the amino acid sequence aa 1-288 of SEQ ID NOs: 1 and 2 or aa 1-335 of SEQ ID NOs: 4 and 33. In particular, the ECD of the FGFR3 polypeptide comprises aa 1-335 of SEQ ID NO: 33.

As used herein, the term "FGFR3-related skeletal disease" refers to a skeletal disease caused by abnormally increased activation of FGFR3, eg, by expression of a gain-of-function mutant of FGFR3. The phrase "gain-of-function mutant of FGFR3" refers to a downstream signaling mutant that, in the presence of an FGF ligand, has, e.g. refers to mutants of FGFR3 that exhibit biological activity that triggers. FGFR3-related skeletal diseases can include hereditary or sporadic diseases. Exemplary FGFR3-related skeletal diseases include, but are not limited to, achondroplasia, thanatophoric osteodysplasia type I (TDI), thanatophoric osteodysplasia type II (TDII), severe achondroplasia with delayed and acanthosis nigricans (SADDAN), achondroplasia, and craniosynthesis premature synostosis syndromes (e.g., Muenke syndrome, Crouzon syndrome, and Crouzon dermatoskeletal syndrome); Examples include hearing loss syndrome (CATSHL).

The terms "fibroblast growth factor" and "FGF" refer to the FGF family, which includes structurally related signaling molecules involved in various metabolic processes, including endocrine signaling pathways, development, wound healing, and angiogenesis. refers to a member. FGFs play important roles in the proliferation and differentiation of a wide range of cell and tissue types. The term preferably refers to FGF1, FGF2, FGF9, FGF18, FGF19, and FGF21, which have been shown to bind to FGFR3. For example, FGF includes human FGF1 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 13), human FGF2 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 14), human FGF9 (e.g., the amino acid sequence of SEQ ID NO: 15) human FGF18 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 38), human FGF19 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 38), and human FGF21 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 39). A polypeptide having the amino acid sequence of

As used herein, the term "fibroblast growth factor receptor 3", "FGFR3" or "FGFR3 receptor" refers to one or more FGFs (e.g., FGF1, FGF2, FGF9, FGF18, FGF19, and/or or FGF21). The human FGFR3 gene is located on the distal short arm of chromosome 4, contains 19 exons, and is an 806 amino acid protein precursor (fibroblast growth factor receptor 3 isoform 1 precursor) containing a signal peptide. ) (eg, a polypeptide having the amino acid sequence of SEQ ID NO: 6 or 35). Mutations in the FGFR3 amino acid sequence that result in skeletal growth disorders (eg, achondroplasia) include, for example, substitution of a glycine residue at position 380 with an arginine residue (ie, G380R). The naturally occurring human FGFR3 gene has the nucleotide sequence shown in Genbank Accession No. NM_000142.4, and the naturally occurring human FGFR3 protein has the amino acid sequence shown in Genbank Accession No. NP_000133, herein referred to as SEQ ID NO: 5. expressed. Wild-type FGFR3 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5) has an extracellular immunoglobulin-like membrane domain (amino acid residues 1-335) including an Ig-like C2-type domain 1-3, a transmembrane domain (amino acid residues (residues 345-377), and an intracellular domain (amino acid residues 378-784). FGFR3 may include fragments and/or variants (e.g., splice variants, e.g., utilizing alternative exon 8 instead of exon 9) of full-length wild-type FGFR3 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5). can be mentioned.

The terms "fragment" and "portion" refer to a portion of a whole, e.g., preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70% of the total length of the reference nucleic acid molecule or polypeptide. , 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. The fragments or portions are, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, It may contain 380, 390, 400, 500, 600, 700 or more amino acid residues, up to the entire length of the reference polypeptide (eg, a polypeptide having the amino acid sequence of SEQ ID NO: 5 or 32). For example, the FGFR3 fragment is at least 200, 205, 210, 215, 220, 225, 235, 230, 240, 245, 250, 255, 260, 265, 275, 280, 285, 290 of SEQ ID NO: 1 or 2, or It can include any polypeptide having 300 contiguous amino acids. Furthermore, the FGFR3 fragment comprises at least 200, 205, 210, 215, 220, 225, 235, 230, 240, 245, 250, 255, 260, 265, 275, 280, 285, 290, 300 of SEQ ID NO: 4 or 33. , 305, 310, 315, 320, 325, 330, 335, 345, or 345 contiguous amino acids.

The term "host cell" as used herein refers to a transporter containing essential cellular components, such as organelles, required for expression of a sFGFR3 polypeptide from a corresponding polynucleotide. Polynucleotide nucleic acid sequences can typically be introduced into host cells by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection). Contained in a nucleic acid vector (eg, a plasmid, artificial chromosome, viral vector, or phage vector). The host cell can be a prokaryotic cell, such as a bacterial or archaeal cell, or a eukaryotic cell, such as a mammalian cell, such as a Chinese hamster ovary (CHO) cell or human embryonic kidney 293 (HEK293). Preferably, the host cell is a mammalian cell, such as a CHO cell.

"Isolated" means separated, recovered, or purified from its natural environment. For example, an isolated sFGFR3 polypeptide (e.g., a sFGFR3 polypeptide or a variant thereof, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2 or 4) may be isolated to some extent after isolation of the sFGFR3 polypeptide from, e.g., a cell culture medium. It can be characterized by its purity. The isolated sFGFR3 polypeptide is present in the preparation at least 75% by weight of the total materials (e.g., polypeptides, polynucleotides, cellular debris, and environmental contaminants) in which the sFGFR3 polypeptide is present in the preparation. constitute at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99% or at least 99.5% by weight of the total material may be at least 75% pure. Similarly, an isolated polynucleotide encoding an sFGFR3 polypeptide (e.g., a polynucleotide having the nucleic acid sequence of SEQ ID NO: 20, 21, 36, or 37) or an isolated host cell (e.g., CHO cells, HEK293 cells, L cells, C127 cells, 3T3 cells, BHK cells, or COS-7 cells), the polynucleotides or host cells are free from any total materials present in the preparation (e.g., polypeptides, polynucleotides, cell debris, and environmental contaminants). ) (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99% by weight of the total materials present in the preparation) % or at least 99.5% by weight).

"Polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and analogs thereof. If present, nucleotide structural modifications can be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after synthesis, for example, by conjugation with a label.

The terms "patient" and "subject" refer to mammals, including, but not limited to, humans (e.g., humans with skeletal growth retardation disorders, e.g., achondroplasia) or non-human mammals (e.g., humans with skeletal growth retardation disorders, e.g., achondroplasia). , a non-human mammal having a skeletal growth retardation disorder, such as achondroplasia), such as a cow, horse, dog, sheep, or cat. Preferably, the patient is a human with a skeletal growth retardation disorder (eg, achondroplasia), particularly an infant, child, or young person with a skeletal growth retardation disorder (eg, achondroplasia).

As used herein, the terms "parenteral administration," "parenterally administered," and other grammatically equivalent phrases generally refer to administration of sFGFR3 polypeptides (e.g., sFGFR3 polypeptides, other than enteral and topical administration by injection). polypeptide or a variant thereof, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or an sFGFR3 polypeptide comprising a signal peptide, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34). Refers to the mode of administration of the composition, including, but not limited to, subcutaneous, intradermal, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, and intracapsular. , intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, intraarticular, subcapsular, intrathecal, intraspinal, epidural, intracerebral, intracranial, intracarotid, and sternal. intravenous injections and infusions.

A "pharmaceutically acceptable excipient, carrier, or adjuvant" is, respectively, one that is physiologically acceptable to a subject (e.g., a human) while also being administered with a pharmaceutical composition (e.g., sFGFR3 Treatment of a polypeptide or a variant thereof, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or an sFGFR3 polypeptide comprising a signal peptide, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34) Refers to a diluent, excipient, carrier, or adjuvant that retains properties. One exemplary pharmaceutically acceptable carrier is saline. Other physiologically acceptable diluents, excipients, carriers, or adjuvants and formulations thereof are known to those skilled in the art.

A "pharmaceutical composition" includes an active agent, e.g., sFGFR3 (e.g., an sFGFR3 polypeptide or a variant thereof, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3 polypeptide comprising a signal peptide); For example, a polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34) in combination with at least one pharmaceutically acceptable excipient, carrier, or diluent. The pharmaceutical composition is for the treatment of a disease or event (e.g., a skeletal growth retardation disorder, e.g., achondroplasia) in a patient (e.g., a patient with a skeletal growth retardation disorder, e.g., achondroplasia). Manufactured or sold as part of a treatment regimen with the approval of a government regulatory agency. Pharmaceutical compositions may be prepared, for example, for parenteral administration, e.g., subcutaneously (e.g., by subcutaneous injection) or intravenously (e.g., in a solvent system suitable for intravenous use, free of particulate embolic material). (as a sterile solution) or for oral administration (eg, as a tablet, capsule, caplet, gelcap, or syrup).

As used herein, the term "sequence identity" refers to a reference sequence, e.g., a wild-type sFGFR3 polypeptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5 or 32) or an sFGFR3 polypeptide (e.g., a sFGFR3 polypeptide or a variant thereof, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a signal peptide) Determine the proportion of amino acid (or nucleic acid) residues in a candidate sequence, e.g. (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignments for the purpose of determining percent identity may be performed using a variety of techniques within the skill of those skilled in the art, such as using publicly available computer software such as BLAST, BLAST-2, BLAST-P, BLAST. -N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, eg, any algorithms needed to achieve maximal alignment over the entire length of the sequences being compared. For example, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence with, about, or to a given reference sequence (some amino acids with, about, or to a given reference sequence) (or a nucleic acid) having or containing percent sequence identity (which can alternatively be expressed as a given candidate sequence) is calculated as follows:
100×(A/B ratio)
(where A is the number of amino acid (or nucleic acid) residues that are scored as identical in the alignment of the candidate and reference sequences, and B is the total number of amino acid (or nucleic acid) residues in the reference sequence. be). In particular, a reference sequence that is aligned for comparison with a candidate sequence is such that the candidate sequence has, for example, 50% to 100% identity over the entire length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence. It can be shown that The length of the candidate sequences that are aligned for comparison purposes is at least 30%, such as at least 40%, such as at least 50%, 60%, 70%, 80%, 90% or 100% of the length of the reference sequence. %. If a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

"Signal peptide" refers to a short peptide (eg, 5-30 amino acids long, eg, 22 amino acids long) at the N-terminus of a polypeptide that directs the polypeptide into the secretory pathway (eg, the extracellular space). The signal peptide is typically cleaved during secretion of the polypeptide. The signal sequence can direct the polypeptide to an intracellular compartment or organelle, such as the Golgi apparatus. Signal sequences can be identified by homology to peptides with known functions that target the polypeptide to specific regions of cells, or by biological activity. Those skilled in the art will be familiar with readily available software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center ter, 1710 University Avenue, Madison, Wis. 53705, BLAST, or the PILEUP/PRETTYBOX program). By this, the signal peptide can be identified. The signal peptide can be, for example, substantially identical to the amino acid sequence of SEQ ID NO: 6 or 35.

The term "skeletal growth retardation disorder" as used herein refers to a skeletal disease characterized by bone deformities and/or dysmorphia. These disorders include, but are not limited to, skeletal growth retardation disorders caused by growth plate (epiphyseal cartilage) fractures, idiopathic skeletal growth retardation disorders, or FGFR3-related skeletal diseases. In particular, patients with skeletal growth retardation disorders (eg, achondroplasia) may have shorter bones than the bones of healthy patients. For example, skeletal growth retardation disorders include osteodysplasias, such as achondroplasia, homozygous achondroplasia, heterozygous achondroplasia, achondroplasia, acral osteogenesis imperfecta, distal Intermediate limb dysplasia, osteogenesis imperfecta, flexor limb dysplasia, chondrodysplasia punctate, chondrodysplasia puncta, cleidocranial dysplasia, congenital shortening of the femur, early craniosynostosis synostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzon dermatoskeletal syndrome), dactyly, brachydactyly, campodactyly, polydactyly, syndactyly, Torsional osteodysplasia, dwarfism, segmental abnormal bone dysplasia, enchondromatosis, fibrochondrogenesis, fibrous osteodysplasia, hereditary multiple exostosis, achondroplasia, Hypophosphatasia, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Kniest osteodysplasia, Kniest syndrome, Langer midlimb dysplasia, Marfan syndrome, McCune-Albright syndrome, microlimb syndrome, metaphyseal abnormalities dysplasia, Jansen's metaphyseal dysplasia, metamorphic dysplasia, Morquio syndrome, Nibelgelt's midlimb dysplasia, neurofibromatosis, osteoarthritis, osteochondral dysplasia, osteogenesis imperfecta, bone Perinatally fatal dysplasia, osteopetrosis, osteoporosis, peripheral osteogenesis imperfecta, Reinhardt syndrome, Roberts syndrome, Robinow syndrome, short rib polydactyly syndrome, short stature, congenital vertebral and epiphyseal dysplasia and vertebral, epiphyseal, and metaphyseal dysplasias.

The terms "soluble fibroblast growth factor receptor 3," "soluble FGFR3," and "sFGFR3" refer to all of the transmembrane domains and any polypeptide moieties that anchor the FGFR3 polypeptide to the cell membrane (e.g., tyrosine kinase domain). Alternatively, it refers to FGFR3 characterized by the absence or functional disruption of essential parts. sFGFR3 polypeptide is a non-membrane bound form of FGFR3 polypeptide. In particular, the transmembrane domain of FGFR3 comprises amino acid residues 345 to 377 of a wild-type FGFR3 sequence (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5) or a wild-type FGFR3 sequence containing a signal peptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 32). extending from amino acid residues 367 to 399 of the polypeptide (having an amino acid sequence). Accordingly, an sFGFR3 polypeptide includes amino acid residues 345-377 of a wild-type FGFR3 polypeptide sequence (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 5) or a wild-type FGFR3 sequence that includes a signal peptide (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 32). Some or all of amino acid residues 367-399 of a polypeptide having an amino acid sequence may be deleted. The sFGFR3 polypeptide comprises the cytoplasmic domain of the wild-type FGFR3 polypeptide sequence (amino acid residues 378-784 of SEQ ID NO: 5) or the cytoplasmic domain of the wild-type FGFR3 polypeptide sequence that includes the signal peptide sequence (amino acid residues 378 of SEQ ID NO: 32). ~806) can be further deleted.

Exemplary sFGFR3 polypeptides include, but are not limited to, at least amino acids 1-100, 1-125, 1-150, 1-175, 1-200, 1-205, 1- 210, 1-215, 1-220, 1-225, 1-230, 1-235, 1-240, 1-245, 1-250, 1-252, 1-255, 1-260, 1-265, 1-270, 1-275, 1-280, 1-285, 1-290, 1-295, or 1-300, or 1-301. The sFGFR3 polypeptides include at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, Any polypeptide having a sequence identity of 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more can be mentioned. Additionally, exemplary sFGFR3 polypeptides include, but are not limited to, at least amino acids 1-100, 1-125, 1-150, 1-175, 1-200, 1-205 of SEQ ID NO: 4 or 33; 1-210, 1-215, 1-220, 1-225, 1-230, 1-235, 1-240, 1-245, 1-250, 1-255, 1-260, 1-265, 1- 270, 1-275, 1-280, 1-285, 1-290, 1-295, 1-300, 1-305, 1-310, 1-315, 1-320, 1-325, 1-330, 1-335, 1-340, 1-345, or 1-348. The sFGFR3 polypeptides include at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%) of any of those sFGFR3 polypeptides having the amino acid sequence of SEQ ID NO: 4 or 33. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity Any polypeptide may be mentioned. Any of the above sFGFR3 polypeptides or variants thereof optionally include a signal peptide in the N-terminal position, e.g., amino acids 1-22 of SEQ ID NO: 6 (MGAPACALALCVAVAIVAGASS) or amino acids 1-19 of SEQ ID NO: 35 (e.g., MMSFVSLLLVGILFHATQA). obtain.

"Treating" and "treatment" refer to the progression or severity of a skeletal growth retardation disorder (e.g., achondroplasia) or skeletal growth in a patient (e.g., human, e.g., infant, child, or young adult). reducing the progression, severity, or frequency (e.g., by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or even 100%). The treatment involves administering the sFGFR3 polypeptide for a period of time (e.g., several days, several The treatment period may be administered for months, years or more. sFGFR3 (e.g., a sFGFR3 polypeptide or a variant thereof, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or a sFGFR3 polypeptide comprising a signal peptide, e.g., having the amino acid sequence of SEQ ID NO: 18 or 34) Exemplary symptoms of achondroplasia that can be treated by polypeptides include, but are not limited to, short stature, long trunk, shortened limbs, adult height of about 42 to about 56 inches, relative a large head, a prominent forehead, an underdeveloped facial area, x-legged legs (e.g., "genu valgum"), an agitated gait, short and thick fingers, short and thick toes, limited ability to extend the arm at the elbow, These include excessive flexion of the lower back, dental disease (eg, from dental crowding), obesity disease, neurological disease, respiratory disease, and/or pain and numbness in the lower back and/or spine.

The term "variant" with respect to a polypeptide refers to a polypeptide that differs by one or more changes in the amino acid sequence from the polypeptide from which the variant is derived (e.g., a parent polypeptide, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 1 or 7). Peptides (e.g., sFGFR3 polypeptides or variants thereof, e.g., polypeptides having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or sFGFR3 polypeptides containing signal peptides, e.g., having the amino acid sequence of SEQ ID NO: 18 or 34) polypeptide). The term "variant" with respect to a polynucleotide refers to a polynucleotide that differs by one or more changes in nucleic acid sequence from the polynucleotide from which the variant is derived (e.g., a parent polynucleotide) (e.g., a polynucleotide encoding an sFGFR3 polypeptide, e.g., A polynucleotide having the nucleic acid sequence of SEQ ID NO: 20, 21, 36, or 37). Changes in the amino acid or nucleic acid sequence of a variant can be, for example, amino acid or nucleic acid substitutions, insertions, deletions, N-terminal truncation, or C-terminal truncation, or any combination thereof. In particular, amino acid substitutions may be conservative and/or non-conservative substitutions. Variants each include a parent polypeptide (e.g., an sFGFR3 polypeptide or a variant thereof, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or an sFGFR3 polypeptide comprising a signal peptide, e.g., SEQ ID NO: 18 or 34 amino acid sequence) or a parent polynucleotide (e.g., a polynucleotide encoding an sFGFR3 polypeptide, e.g., a polynucleotide having a nucleic acid sequence of SEQ ID NO: 20, 21, 36, or 37) can be characterized by sex or nucleic acid sequence identity. For example, a variant may include a polypeptide having an amino acid sequence of SEQ ID NO: 1, 2, 4, or 33 and at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity Any polypeptide may be mentioned. Variants include at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). Mention may also be made of polynucleotides.

A "vector" refers to a sFGFR3 polypeptide (e.g., a sFGFR3 polypeptide or a variant thereof, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, or 33, or an sFGFR3 polypeptide comprising a signal peptide, e.g., SEQ ID NO: 18). or a polypeptide having a 34 amino acid sequence), or a fragment thereof. The vector can be used to infect a cell (e.g., a host cell or a cell of a patient with a human skeletal growth retardation disorder, e.g., achondroplasia) resulting in translation of the vector's polynucleotide into an sFGFR3 polypeptide. can. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell, and are thereby replicated along with the host genome. Additionally, certain vectors are capable of directing the expression of genes to which they are operably linked. Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids.

The term "unit dosage form" means that each unit contains a predetermined amount of active material calculated to produce the desired therapeutic effect in association with any suitable pharmaceutical excipient, carrier, or diluent. Refers to physically distinct units suitable as unit doses for human subjects and other mammals.

References herein to numerical ranges by endpoints are intended to include all numbers subsumed within that range (for example, a reference to 1 to 5 refers to 1, 1.5, 2, 2.75, 3, 3, . . . ). 80, 4, and 5).

Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

2 is a graph showing a sensorgram of sFGFR3 polypeptide. Sensorgrams were analyzed for sFGFR3_Del1 (SEQ ID NO: 7), sFGFR3_Del4 (SEQ ID NO: 1), and sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10; Figure 1A); sFGFR3_Del1 (SEQ ID NO: 7) and sFGFR3_Del1-D3 (SEQ ID NO: 9; Figure 1B). ); sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10), sFGFR3_Del4-LK1-LK2-C253S (SEQ ID NO: 11), and sFGFR3_Del4-LK1-LK2-D3 (SEQ ID NO: 12; Figure 1C); and sFGFR3_Del4 (SEQ ID NO: 1) , sFGFR3_Del4-C253S (SEQ ID NO: 2), and sFGFR3_Del4-D3 (SEQ ID NO: 33; FIG. 1D). FIG. 3 is an image of a Western blot of sFGFR3 polypeptide. Western blots under reducing (R) and non-reducing (NR) conditions were performed for sFGFR3_Del1, sFGFR3_Del1-C253S (SEQ ID NO: 8), and sFGFR3_Del1-D3 (Fig. 2A); sFGFR3_Del4-LK1-LK2, sFGFR3_Del4-LK1-LK2-C253S. , and sFGFR3_Del4-LK1-LK2-D3 (Figure 2B); and sFGFR3_Del4, sFGFR3_Del4-C253S, and sFGFR3_Del4-D3 (Figure 2C). FIG. 3A is a graph showing sensorgrams (FIG. 3A) and proliferation assays (FIG. 3B) of sFGFR3_Del4, sFGFR3_Del4-C253S, and sFGFR3_Del4-D3 using Fgfr3 ^ach/+ chondrocytes in the presence of FGF2. Figure 2 is a graph showing luciferase signaling in serum response element-luciferase (SRE-Luc) HEK cells expressing FGFR3 ^G380R incubated with sFGFR3_Del4-D3 at 0 nM, 70 nM, and 280 nM with or without 1 ng/mL hFGF2. ( ^* indicates p-value < 0.05 compared to sFGFR3_Del4-D3 at 0 nM; ^*** indicates p-value < 0.001). Figure 3 is a graph showing the percentage of surviving animals (wild type (wt) and Fgfr3 ^ach/+ mice) 3 days after treatment with a low dose (0.25 mg/kg) of sFGFR3_Del4-D3 versus age (days). The percentage of surviving wt mice that received vehicle (PBS) is also shown. Ig-like C2-type domains 1 (IgI), 2 (IgII), and variants of wild-type FGFR3 polypeptide (SEQ ID NO: 5 or 32), sFGFR3_Del4-C253S (SEQ ID NO: 2), and sFGFR3_Del4-D3 (SEQ ID NO: 33); 3 is an image showing the amino acid residues corresponding to IgIII. sFGFR3_Del4-C253S contains an amino acid substitution of a cysteine residue by a serine residue at position 253 of SEQ ID NO:1. FIG. 3 is an image of a Western blot of sFGFR3 polypeptide. Western blots under reducing (R) and non-reducing (NR) conditions were performed with 2.3 mg/ml and 23 mg/ml sFGFR3_Del1-D3 (Fig. 7A) and 1.5 mg/ml and 15 mg/ml sFGFR3_Del1-C253S (Fig. 7B) is shown. The melting temperature (T _m ) of sFGFR3_Del4-C253S in 20mM phosphate, 40mM NaCl, pH 7.5 buffer and 40mM citrate, 40mM NaCl, pH 6.5 buffer (Fig. 8A) and 20mM FIG. 8B is a graph showing the T _m of sFGFR3_Del4-D3 in phosphate, 40 mM NaCl, pH 7.5 buffer and 20 mM citrate, 40 mM NaCl, pH 6.5 buffer (FIG. 8B). Figure 2 is a graph showing the fast protein liquid chromatography (FPLC) elution profile of sFGFR3_Del4-D3. FPLC elution profiles are shown in Figure 9A: sFGFR3_Del4-D3, cpm/fraction at 0 min, 2 hours, and 24 hours (Figure 9A); Figure 9B: at 1 minute, 15 minutes, 30 minutes, 2 hours, and 24 hours. sFGFR3_Del4-D3 administered by intravenous bolus, cpm/fraction and normalized to maximum peak (shown in Figure 9B (continued)); Figure 9C: subcutaneously at 30 minutes, 2 hours, 4 hours, and 24 hours. sFGFR3_Del4-D3 administered by injection, cpm/fraction and normalized to maximum peak (shown in Figure 9C (continued)). FIG. 2 is a graph showing the percentage proliferation of Fgfr3 ^ach/+ chondrocytes in the presence of sFGFR3 polypeptide. FIG. Fgfr3 ^ach/+ chondrocyte proliferation was stimulated by sFGFR3_Del4-D3 at 1 ug/ml, 10 ug/ml, and 50 ug/ml (Fig. 10A) and sFGFR3_Del4-C253S at 1 ug/ml, 10 ug/ml, and 50 ug/ml (Fig. 10B). Show about. Figure 2 is a graph showing the PK profile of 2.5 mg/kg sFGFR3_Del4-D3 administered subcutaneously and 2.5 mg/kg sFGFR3_Del4-D3 administered intravenously. Figure 2 is a graph showing the concentration of ¹²⁵ I-sFGFR3_Del4-D3 in kidney, liver, spleen, lung, and heart tissue at 30, 120, and 1440 minutes after intravenous administration. Concentrations are expressed as percent of injected dose per gram (% ID/g). Figure 2 is a graph showing the concentration of ¹²⁵ I-sFGFR3_Del4-D3 in kidney, liver, spleen, lung, and heart tissue at 30, 120, 240, 480, and 1440 minutes after subcutaneous administration. Concentrations are expressed as % ID/g. Figure 2 is a graph showing the concentration (c) and volume of distribution (V _d ) of ¹²⁵ I-sFGFR3_Del4-D3 in brain tissue. c of ¹²⁵ I-sFGFR3_Del4-D3 before and after correction for vascular content and degradation at 30 minutes, 2 hours, and 24 hours after the intravenous bolus (Figure 14A) and at 30 minutes, 2 hours, and 24 hours after the intravenous bolus. Shows the V _d of ¹²⁵ I-sFGFR3_Del4-D3 and RSA after hours (FIG. 14B). Figure 3 is a graph showing the percentage of surviving Fgfr3 ^ach/+ mice that received sFGFR3_Del4-D3. Live wild-type mice, live Fgfr3 ^ach /+ mice, given PBS as vehicle, live Fgfr3 ^ach/+ mice, 2.5 mg/kg sFGFR3_Del4-D3 once weekly, 2.5 mg/kg for 22 days. Live Fgfr3 ach/ ^{+ mice that received /} kg of sFGFR3_Del4-D3 twice a week and live Fgfr3 ach/ ⁺ mice that received 10 mg/kg of sFGFR3_Del4-D3 twice a week are shown. Fgfr3 ^ach administered with PBS as vehicle, 2.5 mg/kg sFGFR3_Del4-D3 once weekly, 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, and 10 mg/kg sFGFR3_Del4-D3 twice weekly. Figure 2 is a graph showing the percentage (%) of locomotor and abdominal respiratory complications in ^/+ mice. FIG. 3 is a graph and an X-ray photograph showing the body length of Fgfr3 ^ach/+ mice administered with sFGFR3_Del4-D3. Wild-type mice administered PBS as vehicle and PBS as vehicle, 2.5 mg/kg sFGFR3_Del4-D3 once weekly, 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, and 10 mg/kg Axial length (FIG. 17A), tail length (FIG. 17B), and tibial length (FIG. 17C) of Fgfr3 ^ach/+ mice that were administered twice weekly with sFGFR3_Del4-D3. Wild-type mice administered PBS as vehicle and Fgfr3 ^{ach/+ mice administered PBS as vehicle, 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, and 10 mg/kg sFGFR3_Del4-D3 twice weekly.} An x-ray of the mouse (Figure 17D) is also shown. All measurements are in millimeters (mm). Figure 3 is a graph showing cranial ratio and an x-ray photograph showing the cranium of Fgfr3 ^ach/+ mice administered with sFGFR3_Del4-D3, respectively. In the graph (FIG. 18A), wild type mice administered PBS as vehicle as well as PBS as vehicle, 2.5 mg/kg sFGFR3_Del4-D3 once weekly, 2.5 mg/kg sFGFR3_Del4-D3 twice weekly Figure 3 shows the cranial ratio (L/W) of Fgfr3 ^ach/+ mice administered twice weekly and 10 mg/kg sFGFR3_Del4-D3 twice a week. In the radiograph (FIG. 18B), wild type mice administered PBS as vehicle, Fgfr3 ^ach/+ mice administered PBS as vehicle, and wild type mice administered 10 mg/kg sFGFR3_Del4-D3 twice weekly. Figure 3 shows the cranium of a Fgfr3 ^ach/+ mouse that received 10 mg/kg of sFGFR3_Del4-D3 twice weekly. Figure 2 is a graph showing real-time kinetic profiles of binding of different concentrations of hFGF1, FGF2, hFGF9, hFGF18, hFGF19, and hFGF21 to immobilized SFGFR3_DEL4-D3. hFGF1 at concentrations from 0.5 nM to 12 nM to immobilized SFGFR3_DEL4-D3 (Figure 19A); hFGF2 at concentrations from 2 nM to 10 nM to immobilized SFGFR3_DEL4-D3 (Figure 19B); 1 nM to 5 nM to immobilized SFGFR3_DEL4-D3 hFGF9 at a concentration of (Figure 19C); hFGF18 at a concentration of 1 nM to 10 nM to immobilized SFGFR3_DEL4-D3 (Figure 19D); hFGF19 at a concentration of 2 nM to 20 nM to immobilized SFGFR3_DEL4-D3 (Figure 19E); and immobilized Kinetic profile for binding of hFGF21 (FIG. 19F) at concentrations from 100 nM to 10000 nM to SFGFR3_DEL4-D3 is shown. The dark overlap line represents the 2:1 binding model used to generate the K _d values. Images of Western blots of retrovirus-infected ATDC5 cells expressing uninduced wild-type ATDC5 and FGFR3 ^G380R . Figure 3 is a graph showing induction of proliferation of ATDC5 FGFR3 ^G380R cells in the presence of SFGFR3_DEL4-D3 for three experiments. Untreated ATDC5 FGFR3 ^G380R cells were used as a control.

We use soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptides and variants thereof to treat skeletal growth retardation disorders in patients (e.g., humans, particularly infants, children, or young adults), e.g. , discovered that achondroplasia can be treated. In particular, sFGFR3 polypeptides of the invention include the following exemplary sFGFR3 polypeptides: sFGFR3_Del4 (sFGFR3_Del4-C253S; SEQ ID NO: 2) containing an amino acid substitution of a cysteine residue by a serine residue at position 253; To provide sFGFR3_Del4 comprising domain 3 (sFGFR3_Del4-D3; SEQ ID NO: 33) as well as variants thereof, e.g., sFGFR3 polypeptides having the amino acid sequence of SEQ ID NO: 4, e.g., amino acids 289-400 of SEQ ID NO: 5 or SEQ ID NO: It is characterized by a deletion of 32 amino acids 311-422. Additionally, the sFGFR3 polypeptide may include a signal peptide, eg, an sFGFR3 polypeptide having the amino acid sequence of SEQ ID NO: 18 or 34. For a description of sFGFR3_Del4 (SEQ ID NO: 1), see U.S. Provisional Patent Application No. 62/276,222 and International Patent Application No. PCT/US16/12553, each of which is incorporated herein by reference in its entirety. (incorporated).

For example, at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, sFGFR3 polypeptides and variants thereof having sequence identity of 95%, 96%, 97%, 98%, 99% or more) may contain amino acid substitutions that remove the cysteine residue at position 253 of SEQ ID NO: 1 (e.g. , sFGFR3_Del4-C253S; polypeptide having the amino acid sequence of SEQ ID NO: 2). In particular, the sFGFR3 polypeptides of the invention may include, for example, the replacement of the cysteine residue at position 253 of SEQ ID NO: 1 by a serine residue. For example, the cysteine residue at position 253 is replaced by a serine residue or another conservative amino acid substitution, such as alanine, glycine, proline, or threonine.

The sFGFR3 polypeptide comprises at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of amino acid residues 23-357 of SEQ ID NO: 32. (sequence identity of)), the polypeptide lacks the signal peptide and transmembrane domain of FGFR3 and is (i) less than 500 amino acids in length; (ii) less than or equal to 200 amino acids in length; and/or (iii) lacks the tyrosine kinase domain of FGFR3 (eg, sFGFR3_Del4-D3; a polypeptide having the amino acid sequence of SEQ ID NO: 33). Also described are methods of administering the sFGFR3 polypeptides of the invention to treat skeletal growth retardation disorders (eg, achondroplasia) in a patient (eg, a human, particularly an infant, child, or young adult).

Described herein are sFGFR3 polypeptides, methods of production, methods of treatment, compositions, and kits of the invention.

Soluble Fibroblast Growth Factor Receptor 3 (sFGFR3) Polypeptides The present invention features sFGFR3 polypeptides and variants thereof that are formulated for the treatment of skeletal growth retardation disorders (eg, achondroplasia). In particular, the sFGFR3 polypeptide has at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%) to the amino acid sequence of SEQ ID NO: 1. , 95%, 96%, 97%, 98%, 99% or more sequence identity), and the sFGFR3 polypeptide contains an amino acid substitution that removes the cysteine residue at position 253 of SEQ ID NO: , sFGFR3_Del4-C253S; polypeptide having the amino acid sequence of SEQ ID NO: 2). For example, the cysteine residue at position 253 of SEQ ID NO: 1 is replaced with a serine residue or a conservative amino acid substitution, such as alanine, glycine, proline, or threonine.

sFGFR3 polypeptides and variants thereof have at least 50% sequence identity to SEQ ID NO: 2 (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) Fragments (e.g., at least amino acids 1-200, 1-205, 1-210, 1-215, 1-220, 1-225, 1-235, 1-230, 1-240, 1-245, 1-250, 1-253, 1-255, 1-260, 1-265, 1-275, 1-280, 1-285, 1-290, or 1-300). Additionally, the sFGFR3 polypeptide can include amino acids 1-301 of SEQ ID NO: 1, and the sFGFR3 polypeptide can include an amino acid substitution of a cysteine residue with a serine residue at position 253 of SEQ ID NO: 1 (e.g., SEQ ID NO: 2). polypeptide having an amino acid sequence).

sFGFR3 polypeptides and variants thereof have at least 50% sequence identity to SEQ ID NO: 33 (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity). (For example, at least amino acids 1-200, 1-210, 1-220, 1-230, 1-240, 1-250, 1-260, 1-270, 1-280, 1-290, 1 ~300, 1-310, 1-320, 1-330, 1-340, 1-340, or 1-345). Additionally, the cysteine residue at position 253 of SEQ ID NO: 4 or 33 and/or position 316 of SEQ ID NO: 4, if present, is replaced by a serine residue or a conservative amino acid substitution, such as alanine, glycine, proline, or threonine. It's good that it has been done.

In view of the results described herein, the invention is not limited to particular sFGFR3 polypeptides or variants thereof. In addition to the exemplary sFGFR3 polypeptides and variants thereof discussed above, the sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (sequence one or more FGFs (e.g., FGF1 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 13)) with similar binding affinity to the sFGFR3 polypeptide (SEQ ID NO: 18 or 34)) containing a signal peptide , FGF2 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 14), FGF9 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 15), FGF18 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 16), FGF19. (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 38), and/or FGF21 (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 39)), for example, skeletal growth retardation disorders, For example, it can be used in a method of treating achondroplasia. The sFGFR3 polypeptide is, for example, a fragment of FGFR3 isoform 2 lacking exons 8 and 9 encoding the C-terminal half of Ig-like C2-type domain 3 and exon 10, which contains the transmembrane domain (e.g., the amino acid sequence of SEQ ID NO: 5 or 32). sequence fragment), which corresponds to a fragment of FGFR3 transcript variant 2 (accession number NM_022965).

The sFGFR3 polypeptides of the invention (eg, sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4)) can include a signal peptide at the N-terminal position. Exemplary signal peptides can include, but are not limited to, amino acids 1-22 of SEQ ID NO: 6 (eg, MGAPACALALCVAVAIVAGASS) or amino acids 1-19 of SEQ ID NO: 35 (eg, MMSFVSLLLVGILFHATQA). Thus, sFGFR3 polypeptides include both secreted forms that lack an N-terminal signal peptide, and non-secreted forms that include an N-terminal signal peptide. For example, a secreted sFGFR3 polypeptide can include the amino acid sequence of SEQ ID NO: 2, 4, or 33. Alternatively, the sFGFR3 polypeptide includes a signal peptide, eg, the amino acid sequence of SEQ ID NO: 18, 19, or 34. Those skilled in the art will appreciate that the position of the N-terminal signal peptide varies in different sFGFR3 polypeptides, e.g., the first 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, It is recognized that it may contain 18, 19, 20, 21, 22, 23, 24, 25, 27, 30 or more amino acid residues. Those skilled in the art can, for example, use appropriate computer algorithms, such as those described by Bendtsen et al. (J. Mol. Biol. 340 (4) 783-795, 2004) and cbs. dtu. The location of the signal sequence cleavage site can be predicted by what is available on the web at dk/services/SignalP/.

Additionally, sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18) or 34)) can be glycosylated. In particular, the sFGFR3 polypeptide can be modified to increase or decrease the extent to which the sFGFR3 polypeptide is glycosylated. Addition or deletion of glycosylation sites to a sFGFR3 polypeptide can be accomplished by altering the amino acid sequence so that one or more glycosylation sites are created or removed. For example, N-linked glycosylation, in which an oligosaccharide is attached to the amide nitrogen of an asparagine residue, results in the amino acid sequences of sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 4 or 33), and their variants. It may occur at positions Asn76, Asn148, Asn169, Asn203, Asn240, Asn272, and/or Asn294. One or more of these Asn residues can also be substituted to eliminate a glycosylation site. For example, O-linked glycosylation, in which an oligosaccharide is attached to the oxygen atom of an amino acid residue, can be performed on sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), variants thereof (SEQ ID NO: 4), and It may occur at positions Ser109, Thr126, Ser199, Ser274, Thr281, Ser298, Ser299, and/or Thr301 of the amino acid sequence of the sFGFR3 polypeptide (SEQ ID NO: 18 or 34), including the signal peptide. Furthermore, O-linked glycosylation can occur at the Ser310 and/or Ser321 positions of sFGFR3_Del4-D3 (SEQ ID NO: 33) and variants thereof (SEQ ID NO: 4). One or more of these Ser or Thr residues can also be substituted to eliminate a glycosylation site.

sFGFR3 Fusion Polypeptides sFGFR3 polypeptides of the invention, such as sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34)) from a heterologous polypeptide (e.g., a fragment crystallizable region (Fc region of an immunoglobulin; e.g., a polypeptide having the amino acid sequences of SEQ ID NOs: 25 and 26) or human serum albumin ( HSA; eg, a polypeptide having the amino acid sequence of SEQ ID NO: 27)) to provide an sFGFR3 fusion polypeptide. Optionally, flexible linkers, such as serine- or glycine-rich sequences (e.g., poly-glycine or poly-glycine/serine linkers, e.g., SEQ ID NOs: 28 and 29), are attached to the sFGFR3 polypeptide and the heterologous polypeptide (e.g., the Fc region or HSA).

For example, sFGFR3 polypeptides such as sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides including a signal peptide (SEQ ID NO: 18 or 34). ) can be a fusion polypeptide comprising, for example, the Fc region of an immunoglobulin in the N-terminal or C-terminal domain. In particular, useful Fc regions include any immunoglobulin molecule from any mammal (e.g., human), including IgG, IgM, IgA, IgD, or IgE and their various subclasses (e.g., IgG- 1, IgG-2, IgG-3, IgG-4, IgA-1, IgA-2). For example, Fc fragment human IgG-1 (SEQ ID NO: 25) or a variant of human IgG-1, such as a variant comprising a substitution of asparagine at position 297 of SEQ ID NO: 25 by alanine (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 26). peptide). Fc fragments of the invention can include, for example, any portion of the heavy chain CH2 and CH3 domains and hinge region. The sFGFR3 fusion polypeptides of the invention may also include, for example, monomeric Fc, eg, CH2 or CH3 domains. The Fc region can optionally be glycosylated at any suitable amino acid residue or residues known to those of skill in the art. The Fc fragments described herein are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, It may have 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50 or more additions, deletions, or substitutions.

Additionally, sFGFR3 polypeptides such as sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34). ) can be conjugated to other molecules at the N-terminal or C-terminal domains for the purpose of improving the solubility and stability of the protein in aqueous solutions. Examples of such molecules include human serum albumin (HSA), PEG, PSA, and bovine serum albumin (BSA). For example, a sFGFR3 polypeptide can be conjugated to human HSA (eg, a polypeptide having the amino acid sequence of SEQ ID NO: 27) or a fragment thereof.

sFGFR3 fusion polypeptides include sFGFR3 polypeptides such as sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34)) and the heterologous polypeptide (eg, Fc region or HSA). The linker region can be of any sequence and length that allows sFGFR3 to remain biologically active, eg, not sterically hindered. Exemplary linker lengths include 1-200 amino acid residues, such as 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101- 110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, or 191-200 amino acid residues. For example, the linker includes or consists of a flexible portion, eg, a region without significant fixed secondary or tertiary structure. Preferred ranges are 5-25 and 10-20 amino acids in length. Such flexibility is generally increased when the amino acids are small and do not have bulky side chains that interfere with rotation or bending of the amino acid chain. Preferably, therefore, the peptide linker of the invention has an increased content of small amino acids, in particular glycine, alanine, serine, threonine, leucine and isoleucine.

Exemplary flexible linkers are glycine-rich, containing, for example, at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even 100% glycine residues. It is a linker. Linkers also include, for example, serine-rich linkers containing at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even 100% serine residues. It is possible. In some cases, the linker amino acid sequence consists only of glycine and serine residues. For example, the linker can be the amino acid sequence of GGGGAGGGG (SEQ ID NO: 28) or GGGGSGGGGSGGGGS (SEQ ID NO: 29). The linker can optionally be glycosylated at any suitable amino acid residue or residues. A linker may be absent, in which case the FGFR3 polypeptide and the heterologous polypeptide (eg, Fc region or HSA) are directly fused together without intervening residues.

Polynucleotides encoding sFGFR3 polypeptides The invention further provides use to treat skeletal growth retardation disorders (e.g., achondroplasia) in patients (e.g., humans, e.g., infants, children, or young adults). sFGFR3 polypeptides that can (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34) ), eg, SEQ ID NO: 20, 21, 36, or 37. For example, the polynucleotide has at least 85% sequence identity (e.g., 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity). Additionally, the polynucleotide has at least 85% sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%) with the nucleic acid sequence of SEQ ID NO: 21 or 37. %, 95%, 96%, 97%, 98%, 99% or more sequence identity)). The invention also provides polynucleotides encoding sFGFR3 fusion polypeptides (e.g., sFGFR3 polypeptides fused to a heterologous polypeptide, e.g., an Fc region or HSA) and sFGFR3 polypeptides without a signal peptide (e.g., sequence 2, 4, and 33) or a sFGFR3 polypeptide with a signal peptide (eg, a polypeptide having the amino acid sequences of SEQ ID NOs: 18, 19, and 34). Additionally, the invention includes polynucleotides that include one or more mutations to alter any of the glycosylation sites described herein.

Optionally, sFGFR3 polynucleotides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34)) in particular the typical codon usage of the host organism (e.g., humans) so as to alter the codons in the nucleic acid without altering the sFGFR3 polypeptide encoded by the nucleic acid sequence of the polynucleotide. Codons can be optimized to reflect A codon-optimized polynucleotide (e.g., a polynucleotide having a nucleic acid sequence of SEQ ID NO: 20, 21, 36, or 37) can be used, e.g., by reducing the GC content and/or in a host cell (e.g., HEK293 cells or CHO). may facilitate genetic manipulation for expression in cells). Codon optimization can be carried out by those skilled in the art, for example by using online tools such as the JAVA Codon Adaptation Tool (www.jcat.de) or the Integrated DNA Technologies Tool (www.eu.idtdna.com/CodonOpt). , poly This can be carried out by simply inputting the nucleic acid sequence of nucleotides and the host organism whose codons are to be optimized. Codon usage frequencies for various organisms can be found in online databases, such as www. Kazusa. or. It is available at jp/codon.

Host Cells for Expression of sFGFR3 Polypeptides Expression of sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or signal peptides) Mammalian cells can be used as host cells for the expression of the sFGFR3 polypeptide (SEQ ID NO: 18 or 34). Exemplary mammalian cell types useful in the present methods include, but are not limited to, human embryonic kidney (HEK; e.g., HEK293) cells, Chinese hamster ovary (CHO) cells, L cells, C127 cells, 3T3 cells, BHK cells, COS-7 cells, HeLa cells, PC3 cells, Vero cells, MC3T3 cells, NS0 cells, Sp2/0 cells, VERY cells, BHK, MDCK cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells , BT20 cells, T47D cells, NSO cells, CRL7O3O cells, and HsS78Bst cells, or any other suitable mammalian host cell known in the art. Alternatively, E. coli cells can be used as host cells for expression of sFGFR3 polypeptides. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® ) 31,537, E. coli BL21 (DE3) (ATCC® BAA-1025), E. coli RV308 (ATCC® 31,608), or as known in the art. Any other suitable E. coli strain may be included.

Vectors Comprising Polynucleotides Encoding sFGFR3 Polypeptides The invention also features recombinant vectors containing any one or more of the polynucleotides described above. The vectors of the invention can be used to generate sFGFR3 polypeptides of the invention, such as sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 containing a signal peptide. Polynucleotides encoding the polypeptide (SEQ ID NO: 18 or 34) can be delivered, including mammalian, viral, and bacterial expression vectors. For example, vectors can be plasmids, artificial chromosomes (eg, BAGs, PACs, and YACs), and viral or phage vectors, and can optionally include promoters, enhancers, or regulatory elements for expression of the polynucleotide. The vector may also contain one or more selectable marker genes, such as ampicillin, neomycin, and/or kanamycin resistance genes for bacterial plasmids, or resistance genes for fungal vectors. The vector can be used in vitro for the production of DNA or RNA, or used to transfect a host cell, e.g., a mammalian host cell, for the production of the sFGFR3 polypeptide encoded by the vector. Alternatively, it can be transformed. Vectors can also be adapted for use in vivo in methods of gene therapy.

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34) ))) include retroviruses, adenoviruses (e.g., Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40). , Ad48, Ad49, Ad50, and Pan9 (also known as AdC68)), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses, e.g., orthomyxoviruses (e.g., influenza viruses), rhabdoviruses ( rabies and vesicular stomatitis viruses), paramyxoviruses (e.g. measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses such as adenoviruses, herpes Viruses (eg, herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (eg, vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding sFGFR3 polypeptides include Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukemia sarcoma, mammalian type C, type B, type D viruses, HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, J.M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B.N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1 996).

Production Methods sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18) or 34)) can be produced by any method known in the art. For example, polynucleotides are produced using molecular cloning methods and placed into vectors, such as plasmids, artificial chromosomes, viral vectors, or phage vectors. The vector is used to transform the polynucleotide into a suitable host cell for expression of the sFGFR3 polypeptide.

Nucleic Acid Vector Construction and Host Cells sFGFR3 polypeptides of the invention, such as sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide. (SEQ ID NO: 18 or 34)) can be produced from host cells. Polynucleotides encoding sFGFR3 polypeptides (e.g., polynucleotides having the nucleic acid sequence of SEQ ID NO: 20, 21, 36, or 37, or variants thereof) can be prepared using conventional techniques known in the art (e.g., transformation, transformation, etc.). The host cell can be contained in a vector that can be introduced into a host cell by transfection, electroporation, calcium phosphate precipitation, direct microinjection, or infection). The choice of vector depends, in part, on the host cell to be used. Generally, host cells can be of either prokaryotic (eg, bacterial) or eukaryotic (eg, mammalian) origin.

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34) )) can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Polynucleotides encoding sFGFR3 polypeptides can be obtained using standard techniques, such as gene synthesis. Alternatively, a polynucleotide encoding a wild-type sFGFR3 polypeptide (e.g., a polypeptide having an amino acid sequence of SEQ ID NO: 5 or 32) can be generated using standard techniques in the art, e.g., QuikChange™ mutagenesis. Defined amino acid substitutions (e.g. serine residues at position 253 of SEQ ID NO: 33 and/or position 316 of SEQ ID NO: 4 or conservative amino acid substitutions, e.g. amino acid substitution of cysteine residues with alanine, glycine, proline, or threonine) can be mutated to contain Polynucleotides encoding sFGFR3 polypeptides can be synthesized using, for example, a nucleotide synthesizer or PCR technology.

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34) )) can be inserted into a vector capable of replicating and expressing the polynucleotide in prokaryotic or eukaryotic host cells. Exemplary vectors useful in this method include, but are not limited to, plasmids, artificial chromosomes, viral vectors, and phage vectors. For example, viral vectors include the aforementioned viral vectors containing a nucleic acid sequence of a polynucleotide encoding an sFGFR3 polypeptide, such as retroviral vectors, adenoviral vectors, or poxvirus vectors (e.g., vaccinia virus vectors, e.g., modified vaccinia Ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors)). Each vector may contain various components that can be adjusted and optimized for compatibility with a particular host cell. For example, vector components include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence of a polynucleotide encoding an sFGFR3 polypeptide, and/or a transcription termination sequence. can be mentioned.

The vectors may be introduced into suitable host cells (e.g., HEK293 cells or CHO cells) using techniques conventional in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. can do. sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34) )) Once the vector has been introduced into a host cell for the production of sFGFR3 polynucleotides (e.g., SEQ ID NOs: 20 and 21 and their variants) in conventional nutrient media modified accordingly. Methods for expressing therapeutic proteins, such as sFGFR3 polypeptides, are known in the art and are described, for example, in Paulina Balbas, Argelia Lawrence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods). in Molecular Biology), Humana Press; 2nd ed. ．． 2004 (July 20, 2004) and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Pres; 2nd ed. 2012 (June 28, 2012), each of which is incorporated herein by reference in its entirety.

sFGFR3 Polypeptide Production, Recovery, and Purification sFGFR3 polypeptides of the invention, including, for example, sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or signal peptides. The host cells (e.g., HEK293 cells or CHO cells) used to produce the sFGFR3 polypeptide (SEQ ID NO: 18 or 34) are known in the art and are grown in a medium suitable for culturing the selected host cells. can be grown. Examples of suitable media for mammalian host cells include minimal essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), Expi293™ expression medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI- 1640 is mentioned. Examples of suitable media for bacterial host cells include Luria broth (LB) and essential supplements such as selective agents such as ampicillin. The host cells are kept at a suitable temperature, such as about 20°C to about 39°C, such as about 25°C to about 37°C, preferably 37°C, and a _CO2 level, such as 5 to 10% (preferably 8 %). The pH of the medium is generally about 6.8-7.4, eg 7.0, depending primarily on the host organism. When an inducible promoter is used in the expression vector, sFGFR3 polypeptide expression is induced under conditions suitable for activation of the promoter.

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34) )) can be recovered from the host cell supernatant. Alternatively, the sFGFR3 polypeptide can be recovered by disrupting the host cells (e.g., using osmotic shock, sonication, or lysis) and then centrifuging or filtering to remove the sFGFR3 polypeptide. . Upon recovery of the sFGFR3 polypeptide, the sFGFR3 polypeptide can be further purified. The sFGFR3 polypeptide can be purified by any method known in the protein purification art, such as protein A affinity, other chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, solubility differences, etc. , or any other standard technique for protein purification (Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009, incorporated herein by reference in its entirety). (see).

Optionally, sFGFR3 polypeptides of the invention such as sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34)) can be conjugated to a detectable label for purification. Examples of suitable labels for use in the purification of sFGFR3 polypeptides include, but are not limited to, protein tags, fluorophores, chromophores, radiolabels, metal colloids, enzymes, or chemiluminescent or bioluminescent molecules. can be mentioned. In particular, protein tags useful for the purification of sFGFR3 polypeptides include, but are not limited to, chromatography tags (e.g., peptide tags consisting of polyanionic amino acids, e.g., FLAG-tags, or hemagglutinin "HA" tags). , affinity tags (e.g., poly(His) tag, chitin binding protein (CBP), maltose binding protein (MBP), or glutathione-S-transferase (GST)), solubilization tags (e.g., thioredoxin (TRX) and poly (NANP)), epitope tags (eg, V5-tag, Myc-tag, and HA-tag), or fluorescent tags (eg, GFP, GFP variants, RFP, and RFP variants).

Methods of Treatment Provided herein are methods of treating skeletal growth retardation disorders in a patient, eg, a patient with achondroplasia (eg, a human with achondroplasia). In particular, the patient is one who exhibits or is likely to develop one or more symptoms of a skeletal growth retardation disorder (eg, achondroplasia). The method comprises administering a sFGFR3 polypeptide of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2)) to a patient with a skeletal growth retardation disorder, such as a patient with achondroplasia (e.g., a human with achondroplasia). , sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34)). In particular, the method provides for administering sFGFR3_Del4-C253S (SEQ ID NO: 2) or sFGFR3_Del4-D3 (SEQ ID NO: 33). For example, the patient is an infant or child with a skeletal growth retardation disorder, eg, an infant, child, or young person with achondroplasia (eg, a human with achondroplasia).

A patient (e.g., a human) can be treated before symptoms of a skeletal growth retardation disorder (e.g., achondroplasia) develop or after symptoms of a skeletal growth retardation disorder (e.g., achondroplasia) develop. . In particular, sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18) or 34)) Patients who can be treated with symptoms such as, but not limited to, shortened limbs, short torso, bowlegs, agitated gait, cranial dysmorphia, cloverleaf cranium, premature craniosynostosis individuals with worm bone, hand deformity, foot deformity, hitchhiker's thumb, and/or thoracic deformity. Additionally, treatment with sFGFR3 polypeptides may result in amelioration of one or more of the above symptoms of skeletal growth retardation disorders, such as achondroplasia (eg, relative to untreated patients).

A patient (e.g., a human) may receive a sFGFR3 polypeptide of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or a signal peptide). The patient may have been diagnosed with a skeletal growth retardation disorder, such as achondroplasia, prior to administration of the polypeptide (SEQ ID NO: 18 or 34). Additionally, patients with skeletal growth retardation disorders, such as achondroplasia, may be those who have not been previously treated with sFGFR3 polypeptides.

Skeletal Growth Retardation Disorders Skeletal growth retardation disorders can be treated by administering sFGFR3 polypeptides described herein to a patient (eg, a human) in need of treatment for a skeletal growth retardation disorder. The method provides for administering sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33)), and variants thereof (SEQ ID NO: 33), to patients (e.g., humans) with skeletal growth retardation disorders. No. 4) or an sFGFR3 polypeptide comprising a signal peptide (SEQ ID No. 18 or 34)). Skeletal growth retardation disorders that can be treated with sFGFR3 polypeptides are characterized by bone deformities and/or malformations and include, but are not limited to, FGFR3-related skeletal diseases. In particular, the patient is treated with sFGFR3_Del4-C253S (SEQ ID NO: 2) or sFGFR3_Del4-D3 (SEQ ID NO: 33).

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34) )) is administered to patients with skeletal growth retardation disorders, including, but not limited to, achondroplasia, achondroplasia, acrodysplasia, distal midlimb dysplasia, and osteogenesis imperfecta. , flexural limb dysplasia, punctate chondrodysplasia, limb root-type chondrodysplasia puncta, clavidocranial dysplasia, congenital shortened femur, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome , Crouzon dermatoskeletal syndrome, finger malformation, brachydactyly, campodactyly, polydactyly, syndactyly, torsion dysplasia, dwarfism, segmental abnormal bone dysplasia, enchondromatosis, fibrosis Chondrogenesis, fibrous osteodysplasia, hereditary multiple exostosis, hypophosphatasia, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Kniest osteodysplasia, Kniest syndrome, Langer's midlimb dysplasia Plasma, Marfan syndrome, McCune-Albright syndrome, microlimb syndrome, metaphyseal dysplasia, Jansen type metaphyseal dysplasia, metamorphic dysplasia, Morquio syndrome, Niebergeld mesodysplasia, neurofibroma osteoarthritis, osteochondrodysplasia, osteogenesis imperfecta, bone Perinatally fatal dysplasia, osteopetrosis, osteoporosis, peripheral osteogenesis imperfecta, Reinhardt syndrome, Roberts syndrome, Robinow syndrome, short rib polydactyly syndrome, short stature, congenital vertebral and epiphyseal dysplasia and vertebral, epiphyseal, and metaphyseal dysplasia.

For example, sFGFR3 polypeptides of the invention (eg, sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18) or 34)) can be used to treat skeletal growth retardation disorders, such as symptoms associated with the above-mentioned disorders, such as achondroplasia. Non-limiting examples of symptoms of skeletal growth retardation disorders that can be treated with sFGFR3 polypeptides include shortened limbs and short torso, bowlegged legs, agitated gait, cranial dysmorphism (e.g., large head), cloverleaf cranium. , craniosynostosis (e.g., premature fusion of bones within the cranium), worm bones (e.g., abnormal filiform connections between bones within the cranium), limb malformations (e.g., polydactyly or extra digits), These include "hitchhiker" thumbs and abnormal fingernails and toenails, and chest deformities (eg, pear-shaped chest or narrow rib cage). Additional conditions that can be treated by administering sFGFR3 polypeptides include non-skeletal abnormalities, such as ocular, oral, and ear malformations, such as congenital cataracts, myopia, in patients with skeletal growth retardation disorders; cleft palate, or hearing loss; brain abnormalities, such as hydrocephalus, porencephaly, hydranencephaly, or corpus callosum defects; heart defects, such as atrial septal defect, patent ductus arteriosus, or transposition of great vessels; Developmental retardation; or mental disorders may also be mentioned.

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34) )) may also increase the survival rate of patients (eg, humans) with skeletal growth retardation disorders (eg, achondroplasia). For example, the survival rate of patients treated with sFGFR3 polypeptides is lower than that of untreated patients with skeletal growth retardation disorders (e.g., achondroplasia) over treatment periods of days, months, years, or more. may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In particular, administration of sFGFR3_Del4-D3 can increase the survival rate (eg, relative to untreated patients) of patients (eg, humans) with skeletal growth retardation disorders, eg, achondroplasia.

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or signal peptides) are administered to a patient (e.g., human). polypeptide (SEQ ID NO: 18 or 34)) that is a FGFR3-associated skeletal disease (e.g., caused by or associated with hyperactivation of FGFR3 as a result of a gain-of-function FGFR3 mutation). Growth retardation disorders such as, but not limited to, achondroplasia, thanatophoric dysplasia type I (TDI), and thanatophoric osteodystrophy can be treated. dysplasia type II (TDII), severe achondroplasia with growth retardation and acanthosis nigricans (SADDAN), achondroplasia, and craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, and Crouzon dermatoskeletal syndrome).

Patients (e.g., humans) with mutations in the FGFR3 gene associated with various FGFR3-related skeletal disorders, such as achondroplasia, achondroplasia, SADDAN, TDI, and TDII, may be treated with the sFGFR3 polypeptides of the invention. (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides including a signal peptide (SEQ ID NO: 18 or 34)). Can be done. For example, sFGFR3 polypeptides can be administered to treat achondroplasia resulting from G380R, G375C, G346E, or S279C mutations in the FGFR3 gene. The following exemplary FGFR3-related skeletal disorders using administration of sFGFR3 polypeptides: Chondrogenesis resulting from G375C, G346E or S279C mutations in the FGFR3 gene; R248C, S248C, G370C, S371C, Y373C, X807R in the FGFR3 gene , X807C, X807G, X807S, X807W and K650M mutations; TDII resulting from the K650E mutation of the FGFR3 gene; and SADDAN resulting from the K650M mutation of the FGFR3 gene.

Any of the above mutations in the FGFR3 gene (e.g., the G380R mutation of the FGFR3 gene) may be present in the sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), or (SEQ ID NO: 4) or a signal peptide (SEQ ID NO: 18 or 34))). can be detected in samples from humans). In addition, the patient's parental and/or fetal samples (e.g., maternal blood, placenta, or fetal nucleic acid obtained from fetal samples) are tested by methods known in the art for mutations in the FGFR3 gene to treat them. Be able to determine the need.

Achondroplasia Achondroplasia is the most common cause of dwarfism in humans and can be treated by administering the sFGFR3 polypeptides described herein. In particular, achondroplasia is associated with sFGFR3 polypeptides of the invention, such as sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or signal peptides. (SEQ ID NO: 18 or 34)). Therefore, administration of sFGFR3 polypeptides can be used to treat symptoms such as, but not limited to, growth retardation, cranial deformity, orthodontic defects, cervical spinal cord compression (e.g., with the risk of death from central apnea or stroke). stenosis of the spinal cord (e.g., pain in the legs and lower back), hydrocephalus (e.g., requiring brain shunt surgery), hearing loss due to chronic otitis, cardiovascular disease, neurological disease, respiratory disease, fatigue. , pain, numbness in the lower back and/or spine, and/or obesity.

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34) Patients treated using )) can include infants, children, and adults with achondroplasia. In particular, infants are often diagnosed with achondroplasia at birth, and therefore treatment with sFGFR3 polypeptides is recommended as early as possible in the patient's life, e.g. immediately after birth, or prenatally (in utero). can be started.

Symptoms of achondroplasia in a patient (e.g., a human) may be caused by the patient using the sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 33)). No. 4) or the sFGFR3 polypeptide containing a signal peptide (SEQ ID No. 18 or 34)) can also be monitored before or after treatment. For example, symptoms of achondroplasia can be monitored prior to treatment to assess the severity and condition of the patient's achondroplasia prior to implementation of the method.

The method includes diagnosing achondroplasia in a patient and monitoring the patient for changes in symptoms of achondroplasia, such as changes in the patient's weight and cranial size (e.g., cranial length and/or cranial width). obtain. Changes in body weight and cranial size occur over a period of time, eg, 1, 2, 3, 4 or more times per month or year, or when the sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4 - D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34)) over the course of treatment with about 1, 2, 3, 4, 5, 6, Monitoring can be done every 7, 8, 12 or 16 weeks. The weight and/or cranial size of a patient with achondroplasia can also be measured at defined events of treatment, eg, before and/or after administration of the sFGFR3 polypeptide.

For example, body weight and/or cranial size can be measured using sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or signal peptides). sFGFR3 polypeptide (SEQ ID NO: 18 or 34)). The body weight of the patient with achondroplasia is measured on a scale, preferably in a standardized manner, e.g. in the same clothing or without clothing, or at certain times of the day, preferably in a fasted state (e.g. breakfast). (the previous morning or at least 1, 2, 3, 4, 5 or more hours after fasting). Cranial size can be described by the length, height, width, and/or circumference of the skull. Measurements can be performed using any known or self-invented standardization method. For human subjects, the measurement of cranial circumference is preferred, which is the maximum possible circumference of the head (e.g. from the most prominent part of the forehead to the maximum width of the occiput) using a flexible, non-stretchable material, e.g. (up to the part) can be wrapped and measured. The cranial height of a subject (eg, a human) can also be measured from the bottom of the chin to the top of the head. Preferably, any measurement is performed more than once, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

Administration of sFGFR3 polypeptides sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide ( SEQ ID NO: 18 or 34)) can be administered by any route known in the art, for example by parenteral, enteral or topical administration. In particular, the sFGFR3 polypeptide can be administered subcutaneously (e.g., by subcutaneous injection), intravenously, intramuscularly, intraarterially, intrathecally, or intraperitoneally to patients with skeletal growth retardation disorders (e.g., achondroplasia). I can do it.

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34) )) can be administered to a patient (e.g., a human) at a predetermined dose, e.g., in an amount effective to treat skeletal growth retardation disorders (e.g., achondroplasia) without inducing significant toxicity. can. For example, the sFGFR3 polypeptide may be administered to a patient with a skeletal growth retardation disorder (eg, achondroplasia) at a dose of about 0.002 mg/kg to about 50 mg/kg (eg, 2.5 mg/kg to 30 mg/kg, 0.5 mg/kg to about 50 mg/kg) 002mg/kg~20mg/kg, 0.01mg/kg~2mg/kg, 0.2mg/kg~20mg/kg, 0.01mg/kg~10mg/kg, 10mg/kg~100mg/kg, 0.1mg/kg kg to 50 mg/kg, 0.5 mg/kg to 20 mg/kg, 1.0 mg/kg to 10 mg/kg, 1.5 mg/kg to 5 mg/kg, or 0.2 mg/kg to 3 mg/kg). Can be administered in individual doses. In particular, sFGFR3 polypeptides can be administered at individual doses of, for example, 0.001 mg/kg to 50 mg/kg, such as 2.5 mg/kg to about 10 mg/kg.

Exemplary doses of sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4 for administration to patients (e.g., humans) with skeletal growth retardation disorders (e.g., achondroplasia) -D3 (SEQ ID NO: 33) and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptide (SEQ ID NO: 18 or 34) containing a signal peptide), for example, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 mg/kg. These doses may be administered one or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more times) per day, week, month, or year. can do. For example, the sFGFR3 polypeptide may be administered to a patient at, for example, about 0.0014 mg/kg/week to about 140 mg/kg/week, such as about 0.14 mg/kg/week to about 105 mg/kg/week, or such as about 1 .4 mg/kg/week to about 70 mg/kg/week (e.g., 2.5 mg/kg/week, 5 mg/kg/week, 10 mg/kg/week, 20 mg/kg/week, 30 mg/kg/week, 40 mg/kg/week kg/week, or 50 mg/kg/week).

Gene Therapy sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18) or 34)) can also be delivered via gene therapy in which a polynucleotide encoding an sFGFR3 polypeptide is delivered to the tissue of interest and expressed in vivo. Methods of gene therapy are described, for example, in Verme et al. (Nature 389:239-242, 1997), Yamamoto et al. (Molecular Therapy 17:S67-S68, 2009), and Yamamoto et al. , (J. Bone Miner. Res. 26:135-142, 2011), each of which is incorporated herein by reference.

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34) )) by cells of a patient (e.g., human) with a skeletal growth retardation disorder (e.g., achondroplasia) in a vector (e.g., plasmid, artificial chromosome) containing a nucleic acid sequence of a polynucleotide encoding an sFGFR3 polypeptide. (eg, BAGs, PACs, and YACs), or viral vectors). For example, the viral vector can be a retroviral vector, an adenoviral vector, or a poxvirus vector (eg, a vaccinia virus vector, eg, modified vaccinia Ankara (MVA)), an adeno-associated virus vector, or an alphavirus vector. The vector can be placed inside the cells of a patient (e.g., human) with a skeletal growth retardation disorder (e.g., achondroplasia), e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, or direct microinjection. This promotes the expression of the sFGFR3 polypeptide, which is then secreted from the cell. The invention further includes cell-based therapies in which cells expressing sFGFR3 polypeptides are administered to a patient (eg, a human).

Pharmaceutical Compositions The pharmaceutical compositions of the present invention contain the sFGFR3 polypeptides of the present invention, such as sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or signal peptides. (SEQ ID NO: 18 or 34)), polynucleotides, vectors, and/or host cells. Compositions containing sFGFR3 polypeptides, polynucleotides, vectors, and/or host cells can be prepared in a variety of formulations, at a range of dosage, and with pharmaceutically acceptable excipients, carriers, or diluents. Can be blended in combination.

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides comprising a signal peptide (SEQ ID NO: 18 or 34) )), polynucleotides, vectors, and/or host cells can be used to treat skeletal growth retardation disorders (e.g., cartilage) in patients (e.g., humans) at prescribed doses, e.g., without inducing significant toxicity. aplasia). For example, the composition may contain about 1 mg/mL to about 500 mg/mL of sFGFR3 polypeptide (eg, 10 mg/mL to 300 mg/mL, 20 mg/mL to 120 mg/mL, 40 mg/mL to 200 mg/mL, 30 mg/mL to sFGFR3 polypeptide).

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34) )), polynucleotides, vectors, and/or host cells, in various forms, e.g., liquid solutions, dispersions or suspensions, powders, or other arrangements suitable for stable storage. structure can be prepared. For example, compositions comprising sFGFR3 polypeptides intended for systemic or local delivery may be suitable for parenteral administration (e.g., subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or intraperitoneal administration). It may be in the form of an injection or infusion solution. sFGFR3 compositions for injection (eg, subcutaneous or intravenous injection) can be formulated using sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, and cell culture media (e.g., Dulbecco's Modified Eagle's Medium (DMEM), α-Modified Eagle's Medium (α-MEM) , F-12 medium). Formulation methods are known in the art and are described, for example, in Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd ed.) Taylor & Fra. ncis Group, CRC Press (2006) (herein incorporated by reference in its entirety) (incorporated into the book).

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34) )), a polynucleotide, a vector, and/or a host cell, a composition comprising a pharmaceutically acceptable excipient, It can be provided in combination with a carrier or diluent. Acceptable excipients, carriers, or diluents can include buffers, antioxidants, preservatives, polymers, amino acids, and carbohydrates. Aqueous excipients, carriers or diluents include water, water-alcoholic solutions, emulsions or suspensions including saline, buffered pharmaceutical parenteral vehicles, such as sodium chloride solution, Ringer's dextrose solution, dextrose and Mention may be made of solutions of sodium chloride, Ringer's solution containing lactose, and fixed oils. Examples of non-aqueous excipients, carriers, or diluents are propylene glycol, polyethylene glycol, vegetable oils, fish oils, and injectable organic esters.

Pharmaceutically acceptable salts also include sFGFR3 polypeptides of the invention, such as sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or signal peptides. sFGFR3 polypeptide (SEQ ID NO: 18 or 34)), a polynucleotide, a vector, and/or a host cell. Exemplary pharmaceutically acceptable salts include mineral salts (e.g., hydrochloride, hydrobromide, phosphate, and sulfate) and salts of organic acids (e.g., acetate, propionate). , malonate, and benzoate). Furthermore, auxiliary substances may be present, such as wetting or emulsifying agents and pH-buffering substances. A detailed discussion of pharmaceutically acceptable excipients, carriers, and diluents can be found in Remington: The Science and Practice of Pharmacy, ^22nd Ed. , Allen (2012) (incorporated herein by reference in its entirety).

sFGFR3 polypeptides of the invention (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 4) or sFGFR3 polypeptides containing a signal peptide (SEQ ID NO: 18 or 34) )), polynucleotides, vectors, and/or host cells can also be formulated with carriers that protect the sFGFR3 polypeptide from rapid release, e.g., controlled release formulations, such as implants and microorganisms. It is an encapsulated delivery system. For example, sFGFR3 compositions may include microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose, gelatin, or poly-(methyl methacrylate) microcapsules; colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, or nanocapsules); or macroemulsions. Additionally, sFGFR3 compositions can be formulated as sustained release compositions. For example, sustained release compositions can include semipermeable matrices of solid hydrophobic polymers containing the sFGFR3 polypeptides, polynucleotides, vectors, or host cells of the invention, where the matrix is formed into a shaped article, e.g., a film or a microorganism. It is in the form of a capsule.

Kits Kits of the invention may contain one or more sFGFR3 polypeptides of the invention described herein (e.g., sFGFR3_Del4-C253S (SEQ ID NO: 2), sFGFR3_Del4-D3 (SEQ ID NO: 33), and variants thereof (SEQ ID NO: 33)). No. 4) or a sFGFR3 polypeptide (SEQ ID No. 18 or 34) containing a signal peptide), polynucleotides, vectors, and/or cells. For example, sFGFR3 polypeptides, polynucleotides, vectors, and/or cells can be administered in liquid form (e.g., water or a buffered salt solution, e.g., 2mM to 20mM sodium phosphate, pH 6.5) in a container (e.g., a glass vial). or 7.0, and 25mM to 250mM sodium chloride). Alternatively, the sFGFR3 polypeptides, polynucleotides, and/or vectors are optionally lyophilized to liquid form in a container (e.g., a glass vial) prior to administration. in lyophilized form, which may contain diluents (eg, water or buffered salt solutions) for reconstitution. The sFGFR3 polypeptides, polynucleotides, vectors, and/or cells may also be present in other formulations described herein in the kit. Kit components may be presented in dosage forms to facilitate administration and may optionally include materials required for administration and/or instructions for treating a patient in accordance with the present methods. For example, a kit can include instructions to guide a user (eg, a physician) regarding administration of sFGFR3 polypeptides, polynucleotides, vectors, and/or cells.

The following examples are intended to illustrate, but not limit, the present disclosure. These studies included the administration of sFGFR3_Del4-C253S (SEQ ID NO: 2) and sFGFR3_Del4-D3 (SEQ ID NO: 33) to patients (e.g., humans) with achondroplasia to treat achondroplasia and its associated symptoms. characterized by administration of an sFGFR3 polypeptide.

Example 1: Production of sFGFR3 polypeptides sFGFR3_Del4-C253S (SEQ ID NO: 2) and sFGFR3_Del4 by transient transfection in three different suspension cell types: HEK293 freestyle, CHO-S freestyle cells and Expi CHO-S cells. -D3 (SEQ ID NO: 33) was produced. For production in HEK293 freestyle and CHO-S freestyle cells, transfections were performed using polyethyleneimine (PEIpro®-Polyplus-transfection) according to the manufacturer's instructions. Protein was collected after 3 days. For sFGFR3 polypeptide production in Expi CHO-S cells, transfections were performed using the High Titer production protocol using Expifectamine as described by the manufacturer. A time course was performed with optimal recovery of sFGFR3 polypeptide after 12 days. Western blots were then performed using 50 ng of sFGFR3 polypeptide. B9 (anti-FGFR3, sc-13121, Santa Cruz) as the primary antibody diluted 1:2000 in blocking buffer and anti-mouse IgG secondary antibody (anti-mouse IgG, diluted 1:5000 in blocking buffer) #7076, Cell signaling). A classic Western blot protocol was used.

Example 2: Purification of sFGFR3 Polypeptides sFGFR3_Del4-C253S and sFGFR3_Del4-D3 were each purified using a two-step purification process involving ion exchange chromatography and size exclusion chromatography.

For ion-exchange chromatography, 300 mL of culture supernatant was removed by cross-flow filtration (AKTA™ flux, GE Healthcare) using 5 μm and 0.2 μm capsules (KGF-A0504TT and KMP-HEC9204TT, respectively, GE Healthcare). Purified. Purified samples containing sFGFR3_Del4-C253S or sFGFR3_Del4-D3 were then loaded at 20 mL/min on the equilibration column (AKTA™ pure25 (GE Healthcare)) after adjustment of the sample conductivity to 14 mS/cm. The column used was HiPrep Q FF26/10 (GE Healthcare) with a bed volume of 53 mL. The binding buffer was 1×PBS and the elution buffer was PBS 1×+1M NaCl. The column was washed with 4 column volumes of 1×PBS. Elution of sFGFR3_Del4-C253S and sFGFR3_Del4-D3 was performed by two steps of 5% NaCl and 10% NaCl using 4 column volumes each. Both 5% NaCl and 10% NaCl were pooled and concentrated by cross-flow filtration (AKTA™ flux, GE Healthcare). The residual volume was then concentrated on a 30 kDa filter by centrifugation (MILLIPORE® UFC903024AMICON® Ultra-15 Centrifugal Filter Concentrator) for 10 minutes at 3,900 g at 4°C. The residual volume was loaded on a HiLoad 26/600 SUPERDEX™ 200 prep grade (28-9893-36, GE Healthcare) with a bed volume of 320 mL for size exclusion chromatography. Loading capacity did not exceed 12.8 mL. Elution was performed in 1×PBS.

Example 3: Kinetic assay and dissociation constant (K _d ) measurement of sFGFR3 polypeptide Calibration-free concentration analysis and kinetic assay of sFGFR3_Del4-C253S and sFGFR3_Del4-D3 were performed using Sensor Chip CM5 (GE Healthcare). Human FGF2 (hFGF2) was covalently immobilized on Sensor Chip CM5 by amine coupling at a level of approximately 5000 RU. To achieve 5000 RU, hFGF2 was immobilized for 420 seconds at a flow rate of 10 μl/min and a concentration of 25 μg/ml. The running buffer was HBS-EP+ buffer (GE Healthcare). The regeneration buffer was 100 mM sodium acetate with 2M sodium chloride, pH 4.5. FGF binding, dissociation constant (K _d ) measurements, and kinetic parameters were measured by surface plasmon resonance using a BIACORE™ T200 (GE Healthcare). The model used for kinetic assays and K _d measurements was a 1:1 binding algorithm.

Example 4: Proliferation assay of sFGFR3 polypeptides Both ATDC5 and ATDC5 FGFR3 ^G380R cell lines were grown on NUNC™ MICROWELL™ 96-Well Optical-Bottom Plates with Polymer Base (ThermoFis 25 in her Scientific, catalog number 165305) The cells were seeded at a density of ,000 cells/ ^cm2 . After a 24-hour incubation period, cells were starved for 48 hours in 0.5% BSA and then stimulated with or without hFGF2 (Peprotech) for 72 hours with sFGFR3_Del4-C253S or sFGFR3_Del4-D3. Cell proliferation was then measured using the CyQUANT® Direct Cell Proliferation Assay (Molecular Probes, Cat. No. C35012). After stimulation, 10 μL of CyQUANT® Direct Cell Proliferation (Invitrogen; 1 ml of 1× PBS, 250 μL of background suppressor, and 50 μL of nuclear stain) was added per well. ATDC5 and ATDC5 FGFR3 ^G380R cells were then incubated for 2 hours at room temperature in the dark. Fluorescence was read using a VARIOSKAN™ LUX multimode microplate reader (ThermoFisher Scientific).

Example 5: Luciferase assay of sFGFR3 polypeptide Serum response element-luciferase ( ^SRE -Luc) HEK cells expressing FGFR3 ^G380R were seeded at a density of 100,000 cells/cm in standard culture 96-well plates. . Cells were then starved for 24 hours with 0.5% heat-inactivated fetal bovine serum (hiFBS) and then stimulated with or without 1 ng/ml hFGF2 by sFGFR3_Del4-D3 at concentrations of 0, 70, and 280 nm. It was treated for 24 hours. Add the culture plate to room temperature for 15 minutes, then add 100 μL per -well Firefly Luc ONE -STEP GLOW ASSAY WORKING SOLUTION (THERMOFISHER SCIEFIC, Catalog Number 16197). , Next, it was shaken for 3 minutes at 600 rpm. Plates were incubated for 10 minutes at room temperature and each cell lysate was transferred to a white opaque 96-well plate to increase luminescence signal and reduce cross-contamination. Luminescent signals were read using a VARIOSKAN™ LUX multimode microplate reader (ThermoFisher Scientific).

Example 6: In Vivo Efficacy Testing of sFGFR3 Polypeptides Experiments were performed on transgenic Fgfr3 ^ach/+ animals in which the expression of mutant FGFR3 was driven by the Col2a1 promoter/enhancer. Mice were exposed to a 12 hour light/dark cycle and had free access to standard laboratory chow and water. The genotype was confirmed by PCR of genomic DNA using primers 5'-AGGTGGCCTTTGACACCTACCAGG-3' (SEQ ID NO: 30) and 5'-TCTGTTGTGTTTCCTCCCTGTTGG-3' (SEQ ID NO: 31), which amplify 360 bp of the FGFR3 transgene.

sFGFR3_Del4-D3 produced using CHO cells was evaluated at twice weekly subcutaneous doses of 0.25 mg/kg. On day 3, all newborn mice from a single liter received the same dose. Control litterers received 10 μl of PBS (vehicle). Thereafter, subcutaneous injections of sFGFR3_Del4-D3 (0.25 mg/kg) were administered twice a week for 3 weeks, alternating on the left and right sides of the back. Mice were observed daily with special attention to changes in locomotion and urination. Breeding was performed to generate litters with half wild type and half heterozygous Fgfr3 ^ach/+ mice. To avoid bias due to phenotypic penetrance variation, experiments were performed on at least two litterers (one treated, one control) from the same breeder. Prior data showed that there were no statistical differences between males and females, therefore males and females were considered as one group for all analyses.

On day 22, all animals were sacrificed by lethal injection of pentobarbital and sexed. All subsequent measurements and analyzes were performed without knowledge of the mouse genotype to avoid investigator bias. Genotyping was performed at the end of the study to clarify the correspondence between the data and the defined genotype. Because achondroplasia is a disease with phenotypic variation, all animals were included in the study. Animals that died before day 22 were used to investigate the effect of treatment on early mortality. Surviving animals at day 22 were used for all analyses. All experiments and data measurements were performed by blinded experimenters at all time points.

Body weights were measured after sacrifice on day 22. The carcasses were carefully skinned, internal organs removed, and skeletal measurements were taken based on X-rays. Organs were collected, weighed, and stored in 10% formalin for further histological analysis using standard paraffin embedding techniques. The organs were then observed for gross abnormalities, such as changes in color or texture and the presence of nodules. During all animal experiments, the Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985; http://grants1.nih.gov/grants/olaw/r reference/phspol.htm) and European commission guidelines for the protection of animals used for scientific purposes (http://ec.europa.eu/environment/chemicals/lab_animals/registration_ en.htm). All procedures were approved by the Institutional Ethic Committee for the use of Laboratory Animals (CIEPAL Azur) (approval number NCE-2012-52).

Example 7: Cell lines used to produce sFGFR3 polypeptides did not affect activity sFGFR3_Del1 (SEQ ID NO: 7), sFGFR3_Del4 (SEQ ID NO: 1) produced in suspended HEK293 cells or CHO cells ), and sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10) were compared for their effects on FGF2 binding activity, Kd, and cell signaling. HEK293 cells or CHO cells differ in post-translational modifications of proteins. Expression of sFGFR3 polypeptide in different cell lines did not affect the Kd of sFGFR3 polypeptide, its binding activity, or its effect on intracellular signaling inhibition (FIGS. 1A-1D).

Example 8: Improving the production of sFGFR3_Del4-C253S and sFGFR3_Del4-D3 The sFGFR3 polypeptides of sFGFR3_Del1 (SEQ ID NO: 7), sFGFR3_Del4 (SEQ ID NO: 1), and sFGFR3_Del4-LK1-LK2 (SEQ ID NO: 10) were modified with serine at position 253. Each was modified to include either an amino acid substitution of a cysteine residue by a residue or an extended Ig-like C2 type domain 3 (SEQ ID NO: 33). These modifications of sFGFR3_Del1 and sFGFR3_Del4-LK1-LK2 had no or minimal effect on the production of sFGFR3 polypeptides as aggregation was still visible (Figures 2A and 2B, respectively). Surprisingly, modification of sFGFR3_Del4 to include either an amino acid substitution of a cysteine residue by a serine residue at position 253 (sFGFR3_Del4-C253S) or an extended Ig-like C2-type domain 3 (SEQ ID NO: 33)) Improved polypeptide production. Notably, there was minimal aggregation of sFGFR3_Del4-C253S and sFGFR3_Del4-D3 under both reducing and non-reducing conditions (Fig. 2C). Inclusion of C253S or D3 also resulted in a relative increase in production compared to sFGFR3_Del4, resulting in a 2-fold increase in sFGFR3_Del4-C253S production and a 3-fold increase in sFGFR3_Del4-D3 production.

Furthermore, sFGFR3_Del4, sFGFR3_Del4-C253S, and sFGFR3_Del4-D3 exhibited similar Kd and were not affected by cell type-specific changes in post-translational modifications. In Expi CHO cells, the Kd of sFGFR3_Del4 was 0.8 nM, the Kd of sFGFR3_Del4-C253S was 0.6 nM, and the Kd of sFGFR3_Del4-D3 was 0.7 nM (Figure 3A and Table 1).

Example 9: sFGFR3_Del4-C253S and sFGFR3_Del4-D3 are equally active in vitro sFGFR3_Del4, sFGFR3_Del4-C253S, and sFGFR3_Del4-D3 are genetically modified to overexpress the FGFR3 ^ach mutation in ATDC5 cells (ATDC5 FGFR3 ^G380R cell line). At a dose of 36 nM, sFGFR3_Del4 produced using HEK293 cells increased proliferation to 115.5%, sFGFR3_Del4 produced using CHO-S cells increased proliferation to 116%, and sFGFR3_Del4 produced using CHO-S cells increased proliferation to 116%. sFGFR3_Del4-C253S produced using CHO-S cells increased proliferation to 114.4%, and sFGFR3_Del4-D3 produced using CHO-S cells increased proliferation to 120.1% (Figure 3B ).

sFGFR3_Del4-D3 was also tested in the FGFR3 ^G380R- expressing SRE(-Luc) HEK cell line at doses of 0 nM, 70 nM, and 280 nM with or without 1 ng/ml hFGF2 (Figure 4; n=8 ). The data shown in Figure 4 are mean +/- standard error of the mean (SEM). These data follow a normal distribution and have equal variance based on the D'Agostino-Pearson omnibus normality test. Statistical comparisons with and without sFGFR3_Del4-D3 were performed using Student's t-test. As shown in Figure 4, sFGFR3_Del4-D3 reduces luciferase signaling in SRE cell lines.

Example 10: sFGFR3_Del4-D3 restores bone growth, prevents mortality, and restores foramen magnum shape in mice with achondroplasia Low dose (0.25 mg/kg) of sFGFR3_Del4-D3 is used In vivo efficacy testing was performed as in Example 6. A total of 60 mice were included in the vehicle group: 32 wild type (wt) mice and 28 Fgfr3 ^ach/+ mice. The treatment group included 40 mice: 19 wt mice and 21 Fgfr3 ^ach/+ mice. Surprisingly, low doses of sFGFR3_Del4-D3 almost completely prevented early death in mice with achondroplasia (Figure 5). In the control group, 53.6% of Fgfr3 ^ach/+ mice died before weaning, whereas only 4.8% of mice in the treatment group died before day 22, after treatment with sFGFR3_Del1 at 0.25 mg/kg. 20% of the mice died (Table 2; see also Garcia et al. Sci. Transl. Med. 5:203ra124, 2013, incorporated herein by reference in its entirety).

sFGFR3_Del4-D3 also partially restored bone growth, correcting the initial differences between wt and Fgfr3 ^ach/+ mice regarding the axial and appendicular skeleton (Table 2). In contrast to previous results of treatment with low doses of sFGFR3_Del1, low doses of sFGFR3_Del4-D3 restored normal foramen magnum shape.

Example 11: Treatment of achondroplasia by administration of sFGFR3_Del4-C253S Human patients (e.g., infants, children, adolescents, or adults) suffering from achondroplasia can be treated at a specific dose (e.g., 0.0002 mg). sFGFR3_Del4-C253S (Figure 6; SEQ ID NO: ) can be administered over the course of days, weeks, months, or years. The progression of achondroplasia treated with sFGFR3_Del4-C253S can be monitored by one or more of several established methods. The physician can monitor the patient by direct observation to assess how the symptoms of achondroplasia exhibited by the patient have changed in response to treatment. For example, a physician may monitor changes in a patient's weight, cranial length, and/or cranial width over a period of time, such as 1, 2, 3, 4 or more times per month or per year over the course of treatment with sFGFR3_Del4-C253S. Monitoring can be done every 1, 2, 3, 4, 5, 6, 7, 8, 12, or 16 weeks. The patient's weight and/or cranial size or changes thereof can also be measured at defined events of treatment, eg, before and/or after administration of sFGFR3_Del4-C253S. For example, body weight and/or cranial size are measured in response to administration of sFGFR3_Del4-C253S.

Example 12: Treatment of achondroplasia by administration of sFGFR3_Del4-D3 Furthermore, human patients (e.g., infants, children, adolescents, or adults) suffering from achondroplasia can be treated at specific doses (e.g., 0 sFGFR3_Del4-D3 (SEQ ID NO: 33) by a suitable route (e.g. by subcutaneous injection) sFGFR3 polypeptides can be administered over the course of days, weeks, months, or years. The progression of achondroplasia treated with sFGFR3_Del4-D3 can be monitored by one or more of several established methods. The physician can monitor the patient by direct observation to assess how the symptoms of achondroplasia exhibited by the patient have changed in response to treatment. For example, a physician may monitor changes in a patient's weight, cranial length, and/or cranial width over a period of time, such as 1, 2, 3, 4 or more times per month or per year over the course of treatment with sFGFR3_Del4-D3. Monitoring can be done every 1, 2, 3, 4, 5, 6, 7, 8, 12, or 16 weeks. The patient's weight and/or cranial size or changes thereof can also be measured at defined events of treatment, eg, before and/or after administration of sFGFR3_Del4-D3. For example, body weight and/or cranial size are measured in response to administration of sFGFR3_Del4-D3.

Example 13: Production of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S sFGFR3_Del4-D3 and sFGFR3_Del4-C253S polypeptides were purified as described in Example 2. Modification of sFGFR3_Del4 to include either an extended Ig-like C2-type domain 3 (FGFR3_Del4-D3) or an amino acid substitution of a cysteine residue with a serine residue at position 253 (sFGFR3_Del4-C253S) improves production of sFGFR3 polypeptides did. In particular, there is less than approximately 2% aggregation of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S under both reducing and non-reducing conditions (SDS-PAGE; Figures 7A and 7B, respectively) using sodium dodecyl sulfate polyacrylamide gel electrophoresis. (observed during loading using concentrations of 2.3 mg/ml or 23 mg/ml for FGFR3_Del4-D3 and 1.5 mg/ml and 15 mg/ml for sFGFR3_Del4-C253S). After the production of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S in fed-batch culture, the top five clones were separated using capillary electrophoresis to yield 0.93-1.0 g/L and 0.98-1.1 g/L. sFGFR3_Del4-D3 and sFGFR3_Del4-C253S were obtained, respectively. Virus filtration using ion exchange chromatography resulted in >60% yield for both sFGFR3_Del4-D3 and sFGFR3_Del4-C253S.

Example 14: Pharmacokinetics and tissue distribution of sFGFR3_Del4-D3 in vivo In vivo studies were conducted to determine the pharmacokinetic parameters of sFGFR3_Del4-D3, the uptake of sFGFR3_Del4-D3 across the blood-brain barrier, and the kidney, liver, spleen, lung, and the tissue distribution of sFGFR3_Del4-D3 in the heart. The study described herein included four treatment groups, with five groups of C57BL/6J mice per treatment group and a total of four mice per group (n=4) (Table 3). Mice were male and weighed 25-30 grams.

Group 1 was sampled at 1 minute, 15 minutes, and 30 minutes; Group 2 was sampled at 4 hours; Group 3 was sampled at 24 hours; Group 4 was sampled at 36 hours. ; Group 5 was sampled at 48 hours. For Group 1, an indwelling intra-arterial catheter (PE-10) was inserted into one common carotid artery under isoflurane anesthesia and used for repeated blood sampling at a final sampling time of 30 minutes. For intravenous injection, ¹²⁵ I-sFGFR3_Del4-D3 was injected intravenously into the jugular vein exposed through a skin incision under isoflurane anesthesia. Group 1 mice remained anesthetized throughout the experiment. Repeat blood samples (2 x approximately 50 μL) were drawn from the arterial catheter 1 and 15 minutes after intravenous injection. For groups 2-5, after injection of ¹²⁵ I-sFGFR3_Del4-D3, the skin was closed with surgical clips and the mice were woken up and returned to their cages. Five minutes before termination time for group 3, mice were re-anesthetized and received an intravenous bolus of ³ H-albumin into the jugular vein. ³ H tracer doses were targeted to obtain a ratio of ¹²⁵ I to ³ H in the blood suitable for dual isotope labeling with lower doses at later sampling times. At the final sampling times (2 hours, 3 hours, 24 hours, 36 hours, and 48 hours), blood samples were collected and animals were euthanized. Brains were sampled for homogenization and measurement of tissue concentration of tracer. Study endpoints included pharmacokinetic parameters (terminal half-life) for sFGFR3_Del4-D3, uptake of sFGFR3_Del4-D3 across the blood-brain barrier, and tissue distribution of sFGFR3_Del4-D3 in the kidney, liver, spleen, lung, and heart. Contained.

Example 15: Thermal and plasma stability of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S Differential scanning calorimetry was used to investigate the thermal stability of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S in mouse plasma. For sFGFR3_Del4-D3, two buffers (20mM phosphate, 40mM NaCl, pH 7.5 and 20mM citrate, 40mM NaCl, pH 6.5) were added to the polypeptide sample. For sFGFR3_Del4-C253S, two buffers (20mM phosphate, 40mM NaCl, pH 7.5, and 40mM citrate, 40mM NaCl, pH 6.5) were added to the polypeptide sample. The melting temperatures (T _m ) for sFGFR3_Del4-C253S in 20mM phosphate, 40mM NaCl, pH 7.5 buffer are 52°C and 56°C and 40mM citrate, 40mM NaCl, pH 6.5 The T _m for sFGFR3_Del4-C253S in buffer was 55°C and 60°C (Figure 8A). For sFGFR3_Del4-D3, two buffers (20mM phosphate, 40mM NaCl, pH 7.5 and 20mM citrate, 40mM NaCl, pH 6.5) were added to the polypeptide sample. The T _m for sFGFR3_Del4-D3 in 20mM phosphate, 40mM NaCl, pH 7.5 buffer is 50°C and 54°C and for sFGFR3_Del4-D3 in 20mM citrate, 40mM NaCl, pH 6.5 buffer. The T _m for sFGFR3_Del4-D3 was 53°C and 58°C (Figure 8B). These results indicate that both sFGFR3_Del4-D3 and sFGFR3_Del4-C253S exhibit two domains of polypeptide stability and unfolding.

Ex vivo plasma stability of sFGFR3_Del4-D3 with a histidine tag was determined by labeling purified sFGFR3_Del4-D3 with a ¹²⁵ I-tracer using the Bolton-Hunter method and then labeling the purified sFGFR3_Del4-D3 with a PD-10 (Sephadex® G-25) column. It was determined by purification as above. Trichloroacetic acid (TCA) precipitability of the peak fractions was also measured to confirm the stability of the ¹²⁵ I-tracer. Mouse plasma (n=4) prewarmed to 37°C was spiked with ¹²⁵ I-sFGFR3_Del4-D3 to a concentration of -10 cpm/mL and then subjected to vortexing. Plasma samples were incubated with ¹²⁵ I-sFGFR3_Del4-D3 in an Eppendorf ThermoMixer® under gentle rotation (300 rpm). Then for TCA precipitation (10 μl sample and 100 μl 2% BSA) and for injection onto a high performance liquid chromatography (FPLC) column (20 μl sample and 150 μl 10 mM PBS, pH 7.4). Aliquots were collected at intervals of , 0, 30, 60, 120, 180, and 360 minutes. Aliquots were stored on ice until TCA precipitation or FPLC injection was performed.

For TCA precipitation, 1 mL of ice-cold 10% TCA was added to the plasma sample, incubated on ice for 10 min, centrifuged at 4,000 g for 5 min, then separated the supernatant and pellet, and both were run in a gamma counter. I counted. For evaluation of ex vivo plasma stability, 100 μl of sample was injected onto a FPLC column (Superdex® 200 10/300GL) and eluted at a rate of 0.75 ml/min for 1.5 column volumes. 1 ml fractions were collected from the column and then counted in a gamma counter. The plasma stability of sFGFR3_Del4-D3 at 37°C was determined to be 95% at 0 minutes, 95% at 2 hours, and approximately 92% at 24 hours, with only slight aggregation (FIG. 9A).

The in vivo stability of sFGFR3_Del4-D3 in plasma after administration by intravenous and subcutaneous injection was also determined. sFGFR3_Del4-D3 was labeled with ¹²⁵ I-tracer using the Bolton-Hunter method and then purified on a PD-10 (Sephadex® G-25) column. ¹²⁵ I-labeled sFGFR3_Del4-D3 (10 μCi in approximately 50 μL of PBS) was administered by intravenous or subcutaneous injection into anesthetized C57Bl/6 mice. ^{The 125} I-tracer protein dose (approximately 0.1 mg/kg) was supplemented with unlabeled protein to a total dose of 2.5 mg/kg. Rat serum albumin, which is used as a vascular marker, was labeled with [ ³ H]-NSP (N-succinidyl [2,3- ³ H] propionate; Perkin Elmer) and labeled with PD-10 (Sephadex® ) G25) Purified on column.

Regarding the stability of sFGFR3_Del4-D3 in plasma after intravenous bolus injection, FPLC elution profile showed no degradation products in plasma up to 15 minutes (Figure 9B). Thirty minutes after administration of sFGFR3_Del4-D3, a small amount of low molecular weight degradation products appeared, which increased by 2 hours, but largely disappeared by 24 hours. Regarding the stability of sFGFR3_Del4-D3 in plasma after subcutaneous injection, the FPLC elution profile showed some degradation products in plasma at 30 minutes, with increased degradation by 2 and 4 hours (Figure 9C). The small amount of tracer remaining in the plasma after 24 hours appears mostly as intact sFGFR3_Del4-D3 polypeptide. The chromatograms in the lower panels for Figures 9B and 9C are presented normalized to the largest peak in each individual run for easier comparison of elution patterns.

Example 16: Ligand binding activity of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S Experiments were performed to characterize the binding affinity of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S for human FGF2. The dissociation constants (Kd) of sFGFR3_Del4-D3 for FGF2 and the Kd of sFGFR3_Del4-C253S were determined as described in Example 3, using a renaturation buffer of 20mM phosphate, 40mM NaCl, pH 7.5. Concentrations of 13 nM, 6.5 nM, 3.25 nM, and 1.75 nM were tested for both sFGFR3_Del4-D3 and sFGFR3_Del4-C253S. The Kd of sFGFR3_Del4-D3 was determined to be approximately 3.6 nm, and the Kd of sFGFR3_Del4-C253S was determined to be approximately 6.9 nm. These results indicate that sFGFR3_Del4-D3 and sFGFR3_Del4-C253S have binding activity for FGF2 in the low nM range.

Example 17: sFGFR3_Del4-D3 and sFGFR3_Del4-C253S exhibit functional activity in vitro A proliferation assay was used to test the functional activity of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S. Proliferation assays using ATDC5 cells genetically modified to overexpress the FGFR3 ^ach mutation (ATDC5 FGFR3 ^G380R cell line) were performed as described in Example 4, with concentrations of 1 ug for sFGFR3_Del4-D3 and sFGFR3_Del4-C253S. /ml, 10ug/ml, and 50ug/ml. At each of these concentrations, sFGFR3_Del4-C253S and sFGFR3_Del4-D3 restored proliferation of FGFR3 ^G380R cells (FIGS. 10A and 10B). The EC50 was determined to be approximately 10 nM for both sFGFR3_Del4-D3 and sFGFR3_Del4-C253S based on a concentration of 1 ug/ml. These results indicate that sFGFR3_Del4-D3 and sFGFR3_Del4-C253S are biologically active in the low nM range.

Example 18: Pharmacokinetic profile of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S Pharmacokinetic (PK) profile of sFGFR3_Del4-D3 administered subcutaneously or intravenously at a dose of 2.5 mg/kg was used to determine the terminal phase of sFGFR3_Del4-D3. The elimination half-life was measured (Figure 11). Samples were collected at 30 minutes, 2 hours, 4 hours, 8 hours, 24 hours, 36 hours and 48 hours for mice that received sFGFR3_Del4-D3 subcutaneously. Samples were collected at 1 minute, 15 minutes, 30 minutes, 2 hours, 24 hours, and 36 hours for mice that received sFGFR3_Del4-D3 intravenously. The subcutaneous terminal elimination half-life of 2.5 mg/kg sFGFR3_Del4-D3 was approximately 20 hours, while the intravenous terminal elimination half-life of 2.5 mg/kg sFGFR3_Del4-D3 was approximately 7 hours. From the PK profile, the T _max was approximately 8 hours, the C _max was approximately 4.5 nM, and the estimated bioavailability was approximately 30% for 2.5 mg/kg sFGFR3_Del4-D3 administered subcutaneously. There was a rapid clearance of intravenously administered sFGFR3_Del4-D3 during the alpha phase followed by a slower beta phase clearance, with a similar intravenous PK profile for sFGFR3_Del4-C253S.

Example 19: Kidney and liver are the main clearance routes for sFGFR3_Del4-D3 Clearance of sFGFR3_Del4-D3 Intravenous administration of 2.5 mg/kg sFGFR3_Del4-D3 in kidney, liver, spleen, lung, and heart tissue and 30, 120, 240, 480, and 1440 minutes after subcutaneous administration of 2.5 mg/kg sFGFR3_Del4-D3. Liver and kidney were the major routes of sFGFR3_Del4-D3 clearance for intravenous administration (Figure 12). Kidney was the major route of sFGFR3_Del4-D3 clearance for subcutaneous administration (Figure 13).

Example 20: sFGFR3_Del4-D3 does not cross the blood-brain barrier Pharmacokinetic studies were also performed to determine the uptake of sFGFR3_Del4-D3 across the blood-brain barrier in wild type mice. Brain tissue uptake of sFGFR3_Del4-D3 was measured at three time points (30 minutes, 2 hours, and 24 hours) after intravenous bolus injection. sFGFR3_Del4-D3 was injected as a radiolabeled tracer ( ¹²⁵ I-sFGFR3_Del4-D3) with 2.5 mg/kg of unlabeled sFGFR3_Del4-D3. The injection dose of ¹²⁵ I-sFGFR3_Del4-D3 is approximately 10 μCi per animal, which corresponds to less than 0.1 mg/kg. After mice were euthanized at 30 minutes, 2 hours, and 24 hours, the concentration of ¹²⁵ I-sFGFR3_Del4-D3 in organs and plasma was measured by liquid scintillation counting.

¹²⁵ I-sFGFR3_Del4-D3 concentrations were corrected for metabolism in plasma and brain samples by measuring the fraction of trichloroacetic acid (TCA) precipitable material (eg, intact tracer). The validity of the TCA correction was also confirmed by injecting the sample onto a size exclusion fast protein liquid chromatography (FPLC) column. Organ concentrations of ¹²⁵ I-sFGFR3_Del4-D3 were corrected for intravascular content (V ₀ ) by injecting radiolabeled albumin ( ³ H-RSA) just before animal sacrifice. The apparent organ distribution volume of RSA represents _V0 . The dose of albumin was negligible (on the order of 1% of physiological concentrations). Concentrations were calculated for all organs except the brain by subtracting the vascular content and considering the TCA precipitable fraction in the plasma. However, no correction was made for the uptake of degraded materials into those organs other than the brain because TCA precipitation was not performed.

The brain concentration is determined by the following formula: C _{brain (corr.)} = [V _d (sFGFR3_Del4-D3) - V ₀ ] x C _{plasma (terminal)} (where V _d (sFGFR3_Del4-D3) is the sFGFR3_Del4 in the brain - the volume of distribution of D3 (calculated as C _brain /C _plasma ), V ₀ is the volume of albumin distributed in the brain, and C _{plasma (terminal)} is the plasma volume of sFGFR3_Del4-D3 at the time of final sample collection. concentration). All concentrations are expressed as percent of the injected dose per gram or ml (% ID/g or % ID/mL), respectively; the dose for intravenous bolus injection is equal to 100%. These values can be converted to [mg/g] or [mg/mL] by multiplying by the injection dose: (body weight, g/1000 g) x 2.5 mg. All body weights ranged from 25g to 30g.

There was no detectable brain uptake of ^125I -sFGFR3_Del4-D3 at any of the measurement time points, as shown by corrected brain concentrations (after correction for vascular content and degradation (TCA precipitability)) (Figure 14A ). Furthermore, the V _d (=V ₀ ) of RSA and ¹²⁵ I-sFGFR3_Del4-D3 were significantly significant at all measurement time points (30 minutes, 2 hours, and 24 hours) as determined by paired t-test. There were no differences (Figure 14B). In summary, there was no measurable uptake of sFGFR3_Del4-D3 into mouse brain tissue at 30 minutes, 2 hours, and 24 hours at a dose of 2.5 mg/kg injected as an intravenous bolus.

Example 21: In vivo efficacy of sFGFR3_Del4-D3 for the treatment of achondroplasia sFGFR3_Del4-D3 and sFGFR3_Del4-C253S at subcutaneous doses of 2.5 mg/kg once or twice weekly or 10 mg/kg twice weekly Each was evaluated. Breeding was performed to generate 30 litterers with half wild type and half heterozygous Fgfr3 ^ach/+ mice (Table 4).

On day 3, all newborn mice from a single liter received the same dose. Control litterers received 10 μl of PBS (vehicle). Thereafter, subcutaneous injections of sFGFR3_Del4-D3 and sFGFR3_Del4-C253S were administered alternately on the left or right side of the back at doses of 2.5 mg/kg once or twice weekly or 10 mg/kg twice weekly for 3 weeks. . Mice were observed daily with particular attention to locomotion and changes in urination and were weighed on the day of injection. Mice with complications were observed twice a day for monitoring. Prior data showed that there were no statistical differences between females and males, therefore females and males were considered as one group for all analyses.

On day 22, all animals were sacrificed by lethal injection of pentobarbital and sexed. All subsequent measurements and analyzes were performed without knowledge of the mouse genotype to avoid investigator bias. Genotyping was performed at the end of the study to clarify the correspondence between the data and the defined genotype. Because achondroplasia is a disease with phenotypic variation, all animals were included in the study. Animals that died before day 22 were used to investigate the effect of treatment on early death. Surviving animals at day 22 were used for all analyses. All experiments and data measurements were performed by blinded experimenters at all time points.

Subcutaneous administration of sFGFR3_Del4-D3 at 2.5 mg/kg once or twice weekly or 10 mg/kg twice weekly significantly reduced survival of Fgfr3 ^ach/+ mice versus Fgfr3 ^ach/+ mice receiving PBS. (Figure 15 and Table 4). Specifically, twice-weekly administration of 10 mg/kg sFGFR3_Del4-D3 resulted in 93% survival of Fgfr3 ^ach/+ mice, whereas once-weekly administration of 2.5 mg/kg sFGFR3_Del4-D3 resulted in Fgfr3 ^{ach/+ mice} twice-weekly administration of 2.5 mg ^/ kg sFGFR3_Del4-D3 resulted in 72% survival of Fgfr3 ^ach/+ mice that received PBS. The survival rate of mice was 62.8%. The mortality rate of Fgfr3 ^ach/+ mice treated with 10 mg/kg sFGFR3_Del4-D3 twice a week was 6.7%, and the mortality rate of mice treated with 2.5 mg/kg sFGFR3_Del4-D3 once a week. was 15.4%, and the mortality rate of Fgfr3 ^ach/+ mice administered with 2.5 mg/kg sFGFR3_Del4-D3 twice weekly was 28.0%, compared with the mortality rate of Fgfr3 ^ach/+ mice administered with PBS. The mortality rate was 37.2%. Statistical analysis of Fgfr3 ^ach/+ mouse survival after treatment with sFGFR3_Del4-D3 was performed using the Agostino-Pearson omnibus normality test followed by a t-test. All study groups passed the normality test. The P values from these analyzes are shown below, where ^* represents a P value of <0.05 and ^*** represents a P value of <0.001 (Table 5).

Subcutaneous administration of sFGFR3_Del4-D3 at 2.5 mg/kg once or twice weekly or 10 mg/kg twice weekly also inhibited locomotor disease and locomotor disease in Fgfr3 ach ^{/ mice versus Fgfr3 ach /+} mice receiving PBS. Reduced the severity and frequency of complications in abdominal breathing (Figure 16). In particular, locomotor disease was most reduced in Fgfr3 ^ach/+ mice that received 10 mg/kg of sFGFR3_Del4-D3 subcutaneously twice weekly, followed by 2.5 mg/kg of sFGFR3_Del4-D3 twice weekly. and mice receiving 2.5 mg/kg of sFGFR3_Del4-D3 once weekly. Complications in abdominal breathing were most reduced in Fgfr3 ^ach/+ mice that received 10 mg/kg of sFGFR3_Del4-D3 subcutaneously twice weekly, followed by 2.5 mg/kg of sFGFR3_Del4-D3 once weekly. sFGFR3_Del4-D3 at 2.5 mg/kg twice weekly. These results indicate that sFGFR3_Del4-D3 reduces the symptoms of achondroplasia in Fgfr3 ^ach/+ mice.

Subcutaneous administration of sFGFR3_Del4-D3 was performed by administering 2.5 mg/kg of sFGFR3_Del4-D3 once or twice a week or 10 mg/kg of sFGFR3_Del4-D3 twice a week to Fgfr3 ^ach/+ mice receiving PBS. Axial length and tail length, as well as total body length including long bones, were also significantly increased in Fgfr3 ^ach/+ mice (p=0.07) (FIGS. 17A-17C). Tail length and body length (axial length) were measured using the same digital caliper on the entire skeleton. Tibial length was measured on digital X-ray. Twice-weekly administration of 10 mg/kg sFGFR3_Del4-D3 resulted in 51% axial correction rate (body length and tail length) in Fgfr3 ^ach/+ mice, followed by twice-weekly administration of 2.5 mg/kg sFGFR3_Del4-D3. This resulted in a 43% axial correction rate for Fgfr3 ^ach/ mice that received and a 39% axial correction rate for Fgfr3 ^ach/+ mice that received 2.5 mg/kg of sFGFR3_Del4-D3 once weekly. Increase in bone length and body length is shown in radiographs of Fgfr3 ^ach/+ mice receiving 2.5 mg/kg or 10 mg/kg of sFGFR3_Del4-D3 twice weekly versus Fgfr3 ^ach/+ mice receiving PBS. It was also clear from (FIG. 17D). Twice-weekly administration of 10 mg/kg sFGFR3_Del4-D3 resulted in 86% appendage correction rate (tibial and femoral length) in Fgfr3 ^ach/+ mice, followed by 2.5 mg/kg sFGFR3_Del4-D3. resulted in a 68% appendage correction rate in Fgfr3 ^ach/+ mice receiving twice weekly and a 54% appendage correction rate in Fgfr3 ^ach/+ mice receiving 2.5 mg/kg sFGFR3_Del4-D3 once weekly. .

Subcutaneous administration of sFGFR3_Del4-D3 also resulted in a dose-dependent improvement in the cranial ratio (length/width (L/W)) of Fgfr3 ^ach/+ mice versus Fgfr3 ^ach/+ mice that received PBS ( Figure 18A). Fgfr3 ^ach/+ mice given subcutaneously 10 mg/kg sFGFR3_Del4-D3 twice weekly exhibited the greatest improvement in head to head ratio (L/W), followed by 2 mg/kg sFGFR3_Del4-D3 twice weekly. Fgfr3 ^ach/+ mice and Fgfr3 ^ach/+ mice treated with 2 mg/kg sFGFR3_Del4-D3 once weekly exhibited improvement. In particular, twice-weekly administration of 10 mg/kg sFGFR3_Del4-D3 resulted in a cranial shape correction rate (L/W ratio) of 37% in Fgfr3 ^ach/+ mice, followed by administration of 2.5 mg/kg sFGFR3_Del4-D3. This resulted in a 29% cranial shape correction rate in Fgfr3 ^ach/+ mice receiving twice weekly and a 19% cranial shape correction rate in Fgfr3 ^ach/+ mice receiving 2.5 mg/kg sFGFR3_Del4-D3 once weekly. Improvement in cranial ratio was also evident in the radiographs of Fgfr3 ^ach/+ mice that received 10 mg/kg sFGFR3_Del4-D3 versus Fgfr3 ^ach/+ mice that received PBS (FIG. 18B). Bone measurements (presented in mm and mean ± SEM) for body length, tail, femur, tibia, and cranial ratio are shown below (Table 6). These results demonstrate dose-dependent in vivo efficacy of sFGFR3_Del4-D3, as evidenced by increased survival, reduced number of complications, increased bone growth, and improved skeletal proportions in Fgfr3 ^ach/+ mice. .

Additionally, a comparison of bone measurements for Fgfr3 ^ach/+ mice administered sFGFR3_Del1 at a twice-weekly dose of 2.5 mg/kg compared to sFGFR3_Del4-D3 at a twice-weekly dose of 2.5 mg/kg. administration was equally or more effective in increasing bone, tail, femur, and tibia length and improving cranial ratio in Fgfr3 ^ach/+ mice (Table 7). Notably, the body length of Fgfr3 ^ach/+ mice receiving sFGFR3_Del4-D3 improved to 135.5 ± 1.75 mm compared to 134.4 ± 1.17 mm for Fgfr3 ^ach/+ mice receiving sFGFR3_Del1. ; the tail length of Fgfr3 ^ach/+ mice receiving sFGFR3_Del4-D3 improved to 73.69 ± 1.5 mm compared to 71.58 ± 0.86 mm for Fgfr3 ^ach/+ mice receiving sFGFR3_Del1; Femoral length of Fgfr3 ^ach/+ mice receiving sFGFR3_Del4-D3 improved to 10.58 ± 0.09 mm compared to 10.01 ± 0.06 mm for Fgfr3 ^ach/+ mice receiving sFGFR3_Del1. ; tibial length of Fgfr3 ^ach/+ mice receiving sFGFR3_Del4-D3 improved to 14.09 ± 0.12 mm compared to 13.27 ± 0.31 mm for Fgfr3 ^ach/+ mice receiving sFGFR3_Del1; ; the cranial ratio of Fgfr3 ^ach/+ mice receiving sFGFR3_Del4-D3 improved to 1.85 ± 0.01 mm compared to 1.81 ± 0.02 mm for Fgfr3 ^ach/+ mice receiving sFGFR3_Del1; .

Example 22: No organ toxicity associated with administration of sFGFR3_Del4-D3 Histopathological studies were performed to characterize organ toxicity associated with administration of sFGFR3_Del4-D3. Wild-type mice (6 males and 6 females per dose) were treated with PBS, 2.5 mg/kg sFGFR3_Del4-D3 once weekly, 2.5 mg/kg sFGFR3_Del4-D3 twice weekly, or 10 mg /kg of sFGFR3_Del4-D3 was administered twice weekly. Organs investigated included kidney, skin, salivary glands, mandibular lymph nodes, gallbladder, spleen, pancreas, lungs, heart, aorta, jejunum, colon, and liver. There were no histopathological results indicative of organ toxicity in wild type mice administered either dose of sFGFR3_Del4-D3. These results demonstrate that there is no toxicity associated with twice weekly administration of sFGFR3_Del4-D3 up to 10 mg/kg.

Example 23: Determination of binding affinity of sFGFR3_Del4-D3 to fibroblast growth factor decided to act as a decoy to disrupt the Surface plasmon resonance was performed using a BIACORE™ T200 (GE Healthcare) to determine the K _d values for different human FGF (hFGF) binding to immobilized sFGFR3_Del4-D3. In particular, K _d values for the paracrine hFGFs hFGF1 (Figure 19A), hFGF2 (Figure 19B), hFGF9 (Figure 19C), and hFGF18 (Figure 19D) and the endocrine hFGFs hFGF19 (Figure 19E) and hFGF21 (Figure 19F) was measured. All four paracrine FGF ligands bound to sFGFR3_Del4-D3 with nanomolar (nM) affinity (Table 8).

Good fits were achieved for FGF2 and FGF18 with the most direct model of binding affinity, the 1:1 binding model. This model accounts for 1:1 binding interactions at the surface of the chip with immobilized SFGFR3_DEL4-D3 binding different FGFs with a single on and off rate: A+B=AB. The 2:1 model also accounts for a 1:1 interaction of FGF binding to SFGFR3_DEL4-D3, but also assumes a conformational change that stabilizes the complex: A+B=AB=AB ^* , resulting in two on and off rates. represents. This model assumes that a conformationally altered complex (SFGFR3_DEL4-D3 bound to FGF) can be dissociated only by reversing the conformational change. We measured experimental data for hFGF1, hFGF9, hFGF19, and hFGF21 to fit the 2:1 model very well, and therefore derived the K _d for hFGF1, hFGF9, hFGF19, and hFGF21 from the 2:1 model. Ta.

Although hFGF1, hFGF9, hFGF19, and hFGF21 all have K _d in the low nM range, their hFGF kinetic profiles were significantly different. For example, FGF1 binds to sFGFR3_Del4-D3 with extremely fast on and off rates, whereas FGF2 does not bind to sFGFR3_Del4-D3 with as fast on or off rates as FGF1, compared to FGF1. Results in an overall lower K _d for FGF2 (Table 8). Significantly lower affinity was measured between sFGFR3_Del4-D3 for paracrine hFGF and hFGF19 or hFGF21, members of the endocrine FGF15/FGF19 subfamily (Table 8 and Figures 19D and 19E). The FGF15/FGF19 subfamily uses Klotho instead of proteoglycans as a cofactor and has evolved into an endocrine-acting growth factor important for the systemic regulation of metabolic parameters, such as phosphate, bile acid, carbohydrate, and lipid metabolism.

These results indicate that there were high affinity interactions between sFGFR3_Del4-D3 and hFGF1, hFGF2, hFGF9, and hFGF18, while low affinity interactions between sFGFR3_Del4-D3 and FGF19 and FGF21 were present. prove that it existed. The low affinity of sFGFR3_Del4-D3 for FGF19 and FGF21 is advantageous. This is because sFGFR3_Del4-D3 has a low probability of interfering with the function of these FGFs in vivo.

Example 24: In vitro proliferation assay of sFGFR3_Del4-D3 After binding of FGF, FGFR3 dimerizes and initiates a signaling cascade. Several downstream signaling pathways are related to FGF signaling. In chondrocytes, dimerized FGFR3 provides anti-proliferative/early differentiation signals to chondrocytes, which ultimately leads to inhibition of bone growth. For example, the RAS / MAPK route propagates a signal for a negative impact on proliferation, final differentiation, and after -split matrix synthesis, and the STAT1 route P107, 130 and cell periodic inhibitory factors P21WAF P21WAF. /Cip1 mediates inhibition of chondrocyte proliferation. Gene expression studies suggest that numerous other pathways are also involved in down-regulating growth-promoting molecules or inducing anti-proliferative functions.

To test the FGFR3 decoy-induced inhibition of FGFR3 ^G380R in a chondrocyte model, and to measure the effect of sFGFR3_Del4-D3 on the proliferation of ATDC5 cells genetically modified to overexpress the FGFR3 ^ach mutation (ATDC5 FGFR3 ^G380R cells). A test was conducted. The chondrocyte cell line ATDC5 cells were first isolated from the differentiated teratocarcinoma stem cell line AT805 and are commonly used as a model for in vitro chondrocyte research. ATDC5 cells were first infected with a retroviral expression vector and a stable cell line expressing FGFR3 ^G380R was generated. Expression of FGFR3 ^G380R in the ATDC5 cell line was measured via Western blot (Figure 20). Extracts of ATDC5 cells expressing FGFR3 ^G380R and control ATDC5 cells at the first passage (G380R#1) and second passage (G380R#2) after resistant cell selection were blotted to determine the total phosphorus content of FGFR3. Oxidation (pFGFR3), defined phosphotyrosine 724 in FGFR3 (pFGFR3 Y724), and total FGFR3 expression (FGFR3) were detected by antibodies. Total extracellular signal-related kinase expression was used as a loading control (ERK). Addition of SFGFR3_DEL4-D3 to ATDC5 FGFR3 ^G380R cells dose-dependently increased the proliferative index of ATDC5 FGFR3 ^G380R cells by 2-fold with an EC ₅₀ of 1.25 +/- 0.27 nM (Figure 21). These results demonstrate that addition of SFGFR3_DEL4-D3 to ATDC5 FGFR3 ^G380R cells overcomes the negative growth signals mediated by FGFR3 ^G380R in a cell model of achondroplasia and induces anti-proliferative effects mediated by FGFR3 in chondrocytes. Consistent with the signal, it demonstrates that chondrocytes are more prominent when expressing FGFR3 containing the G380R mutation.

Embodiments of the invention are further described below:
[Section 1]
A soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptide comprising a polypeptide sequence having at least 90% amino acid sequence identity with amino acid residues 23-357 of SEQ ID NO: 32, the soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptide comprising a signal peptide and a membrane of FGFR3. A polypeptide that lacks a spanning domain and (i) is less than 500 amino acids in length; (ii) comprises an intracellular domain of FGFR3 of no more than 200 contiguous amino acids; or (iii) lacks a tyrosine kinase domain of FGFR3.
[Section 2]
2. The polypeptide of item 1 above, which is less than 475, 450, 425, 400, 375, or 350 amino acids in length.
[Section 3]
2. The polypeptide according to item 1, comprising an intracellular domain of FGFR3 of 175, 150, 125, 100, 75, 50, 40, 30, 20, 15 or less consecutive amino acids.
[Section 4]
According to any one of paragraphs 1 to 3 above, further comprising an amino acid sequence having at least 90%, 92%, 95%, 97% or 99% sequence identity with amino acid residues 423-435 of SEQ ID NO: 32. polypeptide.
[Section 5]
4. The polypeptide according to any one of items 1 to 3 above, further comprising amino acid residues 423 to 435 of SEQ ID NO: 32.
[Section 6]
5, comprising an amino acid sequence having at least 92%, 95%, 97%, 99% or 100% sequence identity with amino acid residues 23-357 of SEQ ID NO: 32. polypeptide.
[Section 7]
6. The polypeptide according to any one of paragraphs 1 to 5 above, comprising an amino acid sequence having at least 92%, 95%, 97%, 99% or 100% sequence identity with SEQ ID NO: 33.
[Section 8]
6. The polypeptide according to any one of paragraphs 1 to 5 above, comprising or consisting of SEQ ID NO: 33.
[Section 9]
A soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptide comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the cysteine residue at position 253 of SEQ ID NO: 1 is removed. A polypeptide further comprising an amino acid substitution.
[Section 10]
10. The polypeptide according to item 9, wherein the cysteine residue at position 253 is replaced with a serine residue.
[Section 11]
11. The polypeptide according to item 9 or 10 above, comprising an amino acid sequence having at least 87%, 90%, 92%, 95%, 97% or 99% sequence identity with the amino acid sequence.
[Section 12]
12. The polypeptide according to any one of the above items 9 to 11, which comprises or consists of the amino acid sequence of SEQ ID NO: 2.
[Section 13]
13. The polypeptide according to any one of paragraphs 1 to 12 above, which is an isolated sFGFR3 polypeptide.
[Section 14]
Fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 18 (FGF18), fibroblast growth factor 19 (FGF19) or 14. The polypeptide according to any one of items 1 to 13 above, which binds to fibroblast growth factor 21 (FGF21).
[Section 15]
15. The polypeptide of paragraph 14 , wherein said binding is characterized by an equilibrium dissociation constant (K _d ) of about 0.2 nM to about 20 nM.
[Section 16]
The binding is characterized by a K _d of about 1 nM to about 10 nM, and optionally, the K _d is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 16. The polypeptide according to item 15 above, which has a wavelength of 9 nm or about 10 nm.
[Section 17]
encoded by a polynucleotide comprising a nucleic acid sequence having at least 85%, 87%, 90%, 92%, 95%, 97% or 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 20, 21, 36 or 37 , the polypeptide according to any one of items 1 to 16 above.
[Section 18]
18. The polypeptide according to item 17, wherein the polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 20, 21, 36 or 37.
[Section 19]
19. The polypeptide according to item 17 or 18 above, wherein the polynucleotide is an isolated polynucleotide.
[Section 20]
A vector comprising the polynucleotide according to any one of items 17 to 19 above.
[Section 21]
21. The vector according to item 20 above, which is selected from the group consisting of plasmids, artificial chromosomes, viral vectors, and phage vectors.
[Section 22]
22. The vector according to item 21 above, which is an isolated polynucleotide.
[Section 23]
A host cell comprising the polynucleotide according to any one of items 17 to 19 above or the vector according to any one of items 20 to 22 above.
[Section 24]
24. The host cell according to item 23 above, which is an isolated host cell.
[Section 25]
25. The host cell according to item 23 or 24 above, which is a HEK293 cell or a CHO cell.
[Section 26]
The polypeptide according to any one of the above items 1 to 16, the polynucleotide according to any one of the above items 17 to 19, the vector according to any one of the above items 20 to 22, or the above item 26. A composition comprising a host cell according to any one of 23 to 25.
[Section 27]
27. The composition according to item 26, further comprising a pharmaceutically acceptable excipient, carrier or diluent.
[Section 28]
28. The composition of paragraph 26 or 27 above, formulated for administration at a dose of about 0.002 mg/kg to about 30 mg/kg.
[Section 29]
29. The composition of paragraph 28, above, formulated for administration at a dose of about 0.001 mg/kg to about 10 mg/kg.
[Section 30]
30. A composition according to any one of paragraphs 26 to 29 above, which is formulated for once-daily, once-weekly, or once-monthly administration.
[Section 31]
Item 26 above, formulated for administration 7 times a week, 6 times a week, 5 times a week, 4 times a week, 3 times a week, 2 times a week, once a week, once every two weeks, or once a month. 31. The composition according to any one of 30 to 30.
[Section 32]
32. The composition of paragraph 31, above, formulated for once-weekly or twice-weekly administration at a dose of about 2.5 mg/kg to about 10 mg/kg.
[Section 33]
33. A composition according to any one of paragraphs 26 to 32 above, which is formulated for parenteral, enteral or topical administration.
[Section 34]
34. The composition according to item 33 above, which is formulated for subcutaneous, intravenous, intramuscular, intraarterial, intrathecal or intraperitoneal administration.
[Section 35]
35. The composition according to item 34, which is formulated for subcutaneous administration.
[Section 36]
A pharmaceutical product comprising the composition according to any one of items 26 to 35 above.
[Section 37]
A method of treating skeletal growth retardation disorder in a patient, the method comprising: the polypeptide according to any one of items 1 to 16 above; the polynucleotide according to any one of items 17 to 19 above; The vector according to any one of paragraphs 22 to 22, the host cell according to any one of paragraphs 23 to 25 above, the composition according to any one of paragraphs 26 to 35 above, or the vector according to any one of paragraphs 36 to 36 above. A method, including administering a medicinal product.
[Section 38]
A method of delivering a polypeptide to a tissue in a patient with a skeletal growth retardation disorder, comprising: an effective amount of the polypeptide according to any one of items 1 to 16 above, or any one of items 17 to 19 above. The polynucleotide according to item 1, the vector according to any one of items 20 to 22 above, the host cell according to any one of items 23 to 25 above, the polynucleotide according to any one of items 26 to 35 above, 37. A method comprising administering the composition according to item 36 or the medicament according to item 36 above.
[Section 39]
39. The method according to item 38, wherein the tissue is skeletal tissue.
[Section 40]
40. The method according to any one of paragraphs 37 to 39, wherein the skeletal growth retardation disorder is a FGFR3-related skeletal disease.
[Section 41]
The FGFR3-related skeletal diseases include achondroplasia, thanatophoric dysplasia type I (TDI), thanatophoric dysplasia type II (TDII), severe achondroplasia with growth retardation and acanthosis nigricans. 41. The method according to item 40, wherein the method is selected from the group consisting of dysplasia (SADDAN), achondroplasia, craniosynostosis premature synostosis syndrome, and syndrome of archdactyly, tall stature, and hearing loss (CATSHL).
[Section 42]
42. The method according to item 41, wherein the skeletal growth retardation disorder is achondroplasia.
[Section 43]
42. The method of item 41, wherein the craniosynostosis premature synostosis syndrome is selected from the group consisting of Muenke syndrome, Crouzon syndrome, and Crouzon dermatoskeletal syndrome.
[Section 44]
44. The method of any one of paragraphs 40 to 43 above, wherein said FGFR3-related skeletal disease is caused by expression of constitutively active FGFR3 in said patient.
[Section 45]
45. The method of paragraph 44, wherein the constitutively active FGFR3 comprises a substitution of a glycine residue by an arginine residue at amino acid position 380 of SEQ ID NO: 5.
[Section 46]
46. The method according to any one of paragraphs 37 to 45 above, wherein the patient has been diagnosed with a skeletal growth retardation disorder.
[Section 47]
The patient has shortened limbs, short torso, bowlegs, agitated gait, cranial dysmorphia, cloverleaf skull, craniosynostosis, worm bones, hand malformations, foot deformities, hitchhiker's thumb, and thoracic deformities. 47. The method of any one of paragraphs 37 to 46 above, wherein the method exhibits one or more symptoms of said skeletal growth retardation disorder selected from the group.
[Section 48]
48. The method of paragraph 47, wherein said patient exhibits improvement in one or more symptoms of said skeletal growth retardation disorder after administration of said polypeptide.
[Section 49]
49. The method of any one of paragraphs 37-48 above, wherein the patient has not been previously administered the polypeptide.
[Section 50]
50. The method of any one of paragraphs 37 to 49 above, wherein the patient is selected from the group consisting of infants, children, young adults, and adults.
[Section 51]
51. The method according to any one of paragraphs 37 to 50 above, wherein the patient is a human.
[Section 52]
52. The method of any one of paragraphs 37-51, above, wherein the polypeptide is administered to the patient at a dose of about 0.002 mg/kg to about 30 mg/kg.
[Section 53]
53. The method of paragraph 52, wherein the polypeptide is administered to the patient at a dose of about 0.001 mg/kg to about 10 mg/kg.
[Section 54]
54. The method according to any one of items 37 to 53, wherein the polypeptide is administered to the patient at least once a day, at least once a week, at least once a month, or at least once a year.
[Section 55]
The above item, wherein the polypeptide is administered to the patient 7 times a week, 6 times a week, 5 times a week, 4 times a week, 3 times a week, twice a week, once a week, once every two weeks, or once a month. 55. The method according to any one of 37 to 54.
[Section 56]
56. The method of paragraph 55, wherein the polypeptide is administered to the patient at a dose of about 0.25 mg/kg to about 20 mg/kg at least about once or more times a week.
[Section 57]
57. The method of paragraph 56, wherein the polypeptide is administered to the patient at a dose of about 2.5 mg/kg or about 10 mg/kg once or twice a week.
[Section 58]
58. The method of any one of paragraphs 37 to 57 above, wherein the polypeptide is administered to the patient in a composition comprising a pharmaceutically acceptable excipient, carrier or diluent.
[Section 59]
59. The method according to item 58, wherein the composition is administered to the patient subcutaneously, intravenously, intramuscularly, intraarterially, intrathecally, or intraperitoneally.
[Section 60]
60. The method according to item 59, wherein the composition is administered to the patient by subcutaneous injection.
[Section 61]
61. The method of paragraph 59 or 60, above, wherein the polypeptide has an in vivo half-life of about 2 hours to about 25 hours.
[Section 62]
62. The method of any one of paragraphs 37-61 above, wherein administration of the polypeptide increases survival of the patient.
[Section 63]
63. The method of any one of paragraphs 37-62, above, wherein administering the polypeptide improves locomotion in the patient.
[Section 64]
64. The method of any one of paragraphs 37-63 above, wherein administration of the polypeptide improves abdominal breathing in the patient.
[Section 65]
65. The method of any one of paragraphs 37 to 64 above, wherein administration of the polypeptide increases body length and/or bone length in the patient.
[Section 66]
66. The method of any one of paragraphs 37 to 65 above, wherein administration of the polypeptide improves cranial ratio and/or restores foramen magnum shape in the patient.
[Section 67]
Cultivating the host cell according to any one of paragraphs 23 to 25 above in a culture medium under conditions suitable for causing expression of the polypeptide, and recovering the polypeptide from the culture medium. 17. The method for producing the polypeptide according to any one of the above items 1 to 16, comprising:
[Section 68]
68. The method of paragraph 67, wherein said collection comprises chromatography.
[Section 69]
69. The method of paragraph 68, wherein the chromatography comprises affinity chromatography or size exclusion chromatography.
[Section 70]
70. The method of item 69, wherein the affinity chromatography comprises ion exchange chromatography or anti-FLAG chromatography.
[Section 71]
71. The method of item 70, wherein the anti-FLAG chromatography comprises immunoprecipitation.
[Section 72]
17. A polypeptide according to any one of paragraphs 1 to 16 above for treating skeletal growth retardation disorders in a patient.
[Section 73]
73. The polypeptide according to item 72, wherein the skeletal growth retardation disorder is a FGFR3-related skeletal disease.
[Section 74]
74. The polypeptide according to item 73, wherein the FGFR3-related skeletal disease is selected from the group consisting of achondroplasia, TDI, TDII, SADDAN, achondroplasia, craniosynostosis syndrome, and CATSHL.
[Section 75]
75. The polypeptide according to item 74, wherein the FGFR3-related skeletal disease is achondroplasia.
[Section 76]
76. The polypeptide according to item 75, wherein the craniosynostosis premature synostosis syndrome is selected from the group consisting of Muenke syndrome, Crouzon syndrome, and Crouzon dermatoskeletal syndrome.
[Section 77]
77. The polypeptide according to any one of paragraphs 73 to 76, wherein said FGFR3-related skeletal disease is caused by expression of constitutively active FGFR3 in said patient.
[Section 78]
78. The polypeptide of paragraph 77, wherein the constitutively active FGFR3 comprises a substitution of a glycine residue by an arginine residue at amino acid position 380 of SEQ ID NO: 5.
[Section 79]
79. The polypeptide according to any one of paragraphs 72 to 78, wherein the patient has been diagnosed with the skeletal growth retardation disorder.
[Section 80]
The patient has shortened limbs, short torso, bowlegs, agitated gait, cranial dysmorphia, cloverleaf skull, craniosynostosis, worm bones, hand malformations, foot deformities, hitchhiker's thumb, and thoracic deformities. 80. The polypeptide according to any one of paragraphs 72 to 79 above, which exhibits one or more symptoms of the skeletal growth retardation disorder selected from the group.
[Section 81]
81. The polypeptide of paragraph 80, wherein said patient exhibits improvement in one or more symptoms of said skeletal growth retardation disorder after administration of said sFGFR3 polypeptide.
[Section 82]
82. The polypeptide of any one of paragraphs 72 to 81 above, wherein said patient has not previously been administered said polypeptide.
[Section 83]
83. The polypeptide according to any one of paragraphs 72 to 82 above, wherein the patient is selected from the group consisting of infants, children, young adults, and adults.
[Section 84]
84. The polypeptide according to any one of paragraphs 72 to 83 above, wherein the patient is a human.
[Section 85]
85. The polypeptide of any one of paragraphs 72 to 84 above, wherein the polypeptide is administered to said patient at a dose of about 0.002 mg/kg to about 30 mg/kg.
[Section 86]
86. The polypeptide of paragraph 85, wherein the polypeptide is administered to said patient at a dose of about 0.001 mg/kg to about 10 mg/kg.
[Section 87]
87. The polypeptide according to any one of paragraphs 72 to 86, which is administered to the patient at least once a day, at least once a week, at least once a month, or at least once a year.
[Section 88]
Items 72 to 87 above, administered to said patient 7 times a week, 6 times a week, 5 times a week, 4 times a week, 3 times a week, 2 times a week, once a week, once every two weeks, or once a month. The polypeptide according to any one of .
[Section 89]
89. The polypeptide of paragraph 88, wherein the polypeptide is administered to said patient twice a week at a dose of about 2.5 mg/kg to about 10 mg/kg.
[Section 90]
90. A polypeptide according to any one of paragraphs 72 to 89 above, which is administered to said patient in a composition comprising a pharmaceutically acceptable excipient, carrier, or diluent.
[Section 91]
91. The polypeptide of paragraph 90, wherein said composition is administered parenterally, enterally, or topically to said patient.
[Section 92]
92. The polypeptide of paragraph 91, wherein the composition is administered to the patient subcutaneously, intravenously, intramuscularly, intraarterially, intrathecally, or intraperitoneally.
[Section 93]
93. The polypeptide of paragraph 92, wherein said composition is administered to said patient by subcutaneous injection.
[Section 94]
Fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), fibroblast growth factor 18 (FGF18), fibroblast growth factor 19 (FGF19), or the polypeptide according to any one of the above items 72 to 93, which binds to fibroblast growth factor 21 (FGF21).
[Section 95]
9. A method for producing the polypeptide according to any one of items 1 to 8 above, which comprises deleting part of the signal peptide, transmembrane domain, and intracellular domain from the FGFR3 polypeptide.
[Section 96]
96. The method according to item 95, wherein the portion of the intracellular domain consists of amino acid residues 436-806 of SEQ ID NO: 32.
[Section 97]
13. A method for producing the polypeptide according to any one of items 9 to 12 above, which comprises introducing an amino acid substitution that removes the cysteine residue at position 253 of SEQ ID NO: 1.
[Section 98]
The polypeptide according to any one of the above items 1 to 16, the polynucleotide according to any one of the above items 17 to 19, the vector according to any one of the above items 20 to 22, or the above item 26. A kit comprising a host cell according to any one of claims 23 to 25, optionally comprising instructions for use of said kit.
All publications, patents, and patent applications mentioned in the above specification are referred to in the same and identical manner as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Incorporated herein by reference. Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it is understood that it may be further modified and the claimed invention should not be unduly limited to such specific embodiments. be done. Indeed, various modifications of the described modes of carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application generally covers any variations, uses, or adaptations of the invention according to its principles, and any such departure from this disclosure is within the range of known and conventional practice within the field to which the invention pertains. and shall be within the scope and applicable to the essential features set forth herein above.

Claims (13)

A kit comprising a soluble fibroblast growth factor receptor 3 (sFGFR3) polypeptide comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 4, the sFGFR3 polypeptide being in liquid form or frozen. A kit, characterized in that it is present in a container in dry form. The sFGFR3 polypeptide is linked to at least one of fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 9 (FGF9), or fibroblast growth factor 18 (FGF18). 2. The kit of claim 1, which binds to the kit. Kit according to claim 1 or 2, wherein the binding is characterized by an equilibrium dissociation constant (Kd) of 0.2 nM to 20 nM. 4. The kit according to any one of claims 1 to 3, wherein the sFGFR3 polypeptide consists of the amino acid sequence of SEQ ID NO: 33. 5. A kit according to any one of claims 1 to 4, wherein the sFGFR3 polypeptide is in liquid form. 5. A kit according to any one of claims 1 to 4, wherein the sFGFR3 polypeptide is in lyophilized form. 7. The kit of any one of claims 1 to 6, wherein the kit further comprises a diluent for reconstitution of the lyophilized sFGFR3 polypeptide. 8. A kit according to claim 7, wherein the diluent is water or a buffered salt solution. 9. A kit according to any one of claims 1 to 8, further comprising instructions for administration. 10. A kit according to any one of claims 1 to 9 for use in the treatment of FGFR3-related skeletal growth retardation disorders. FGFR3-related skeletal growth retardation disorders include achondroplasia, thanatophoric dysplasia type I (TDI), thanatophoric dysplasia type II (TDII), severe cartilage with growth retardation and acanthosis nigricans. 11. The kit of claim 10, wherein the kit is selected from the group consisting of aplasia aplasia (SADDAN), achondroplasia, craniosynostosis, and acanthosis nigricans, tall stature, and hearing loss syndrome (CATSHL). 11. The kit of claim 10, wherein the FGFR3-related skeletal growth retardation disorder is achondroplasia. 11. The kit of claim 10, wherein the FGFR3-related skeletal growth retardation disorder is achondroplasia.